Methods of treating autoimmune disease via CTLA-4Ig

ABSTRACT

The method of immunotherapy of the present invention involves the regulation of the T cell immune response through the activation or suppression/inactivation of the CD28 pathway. Induction of activated T cell lymphokine production occurs upon stimulatory binding of the CD28 surface receptor molecule, even in the presence of conventional immunosuppressants. Inhibition of CD28 receptor binding to an appropriate stimulatory ligand or inactivation of the CD28 signal transduction pathway through other means down-regulates CD28-pathway related T cell lymphokine production and its resulting effects.

RELATED APPLICATIONS

[0001] This is a continuation-in-part of International ApplicationSerial No. PCT/US93/03155, entitled “CD28 Pathway Immunoregulation,”filed Apr. 6, 1993 by Thompson et al., which is a continuation of U.S.application Ser. No. 07/864,805, entitled “CD28 PathwayImmunoregulation,” U.S. application Ser. No. 07/864,807, entitled“Immunotherapy Involving Stimulation of T_(H)CD28 LymphokineProduction,” and U.S. application Ser. 07/864,866, entitled “Enhancementof CD28-Related Immune Response,” all filed Apr. 7, 1992 by Thompson, etal., which are continuations-in-part of U.S. Ser. No. 07/275,433,entitled “Immunotherapy Involving CD28 Stimulation,” filed Nov. 23, 1988by Thompson et al., now abandoned, and is also a continuation-in-part ofInternational Application Serial No. PCT/US89/05304 (Publication No. WO90/05541), entitled “Immunotherapy Involving CD28 Stimulation,” filedNov. 22, 1989 by Thompson et al. and U.S. patent Ser. No. 07/902,467entitled “Immunotherapy Involving CD28 Stimulation,” filed Jun. 19,1992by Thompson et al., all herein incorporated by reference.

SPONSORSHIP

[0002] Work on this invention was supported in part by Naval MedicalResearch and Development Command, Research Task No. M0095.0003-1007. TheGovernment has certain rights in the invention.

BIOLOGICAL DEPOSIT

[0003] Murine hybridoma cell line 9.3 has been deposited with theAmerican Type Culture Collection in Rockville, Md., in compliance withthe provisions of the Budapest Treaty, and has been assigned ATCCDesignation No. HB10271.

FIELD OF THE INVENTION

[0004] The present invention generally relates to immunotherapy. Moreparticularly, the present invention relates to immunotherapy involvingregulation of the CD28 T cell surface molecule.

BACKGROUND OF THE INVENTION

[0005] Thymus derived lymphocytes, referred to as T cells, are importantregulators of in vivo immune responses. T cells are involved incytotoxicity and delayed type hypersensitivity (DTH), and provide helperfunctions for B lymphocyte antibody production. In addition, T cellsproduce a variety of lymphokines which function as immunomodulatorymolecules, such as for example, interleukin-2 (IL-2), which canfacilitate the cell cycle progression of T cells; tumor necrosisfactor-α (TNF-α) and lymphotoxin (LT), cytokines shown to be involved inthe lysis of tumor cells; interferon-γ (IFN-γ), which displays a widevariety of anti-viral and anti-tumor effects; and IL-3 andgranulocyte-macrophage colony stimulating factor (GM-CSF), whichfunction as multilineage hematopoietic factors.

[0006] Current immunotherapeutic treatments for diseases such as cancer,acquired immunodeficiency syndrome (AIDS) and attending infections,involve the systemic administration of lymphokines, such as IL-2 andIFN-γ, in an attempt to enhance the immune response. However, suchtreatment results in non-specific augmentation of the T cell-mediatedimmune response, since the lymphokines administered are not specificallydirected against activated T cells proximate to the site of infection orthe tumor. In addition, systemic infusions of these molecules inpharmacologic doses leads to significant toxicity. Present therapies forimmunodeficient or immunodepressed patients also involve non-specificaugmentation of the immune system using concentrated γ-globulinpreparations. The stimulation of the in vivo secretion ofimmunomodulatory factors has not, until now, been considered a feasiblealternative due to the failure to appreciate the effects and/ormechanism and attending benefits of such therapy.

[0007] It would thus be desirable to provide a method of immunotherapywhich enhances the T cell-mediated immune response and which is directedspecifically toward T cells activated by an antigen produced by thetargeted cell. It would further be desirable to provide a method ofimmunotherapy which could take advantage of the patient's naturalimmunospecificity. It would also be desirable to provide a method ofimmunotherapy which can be used in immunodepressed patients. It wouldadditionally be desirable to provide a method of immunotherapy whichdoes not primarily rely on the administration of immunomodulatorymolecules in amounts having significant toxic effects.

[0008] It would also be desirable to provide a method of immunotherapywhich, if so desired, could be administered directly without removal andreintroduction of T cell populations. It would further be desirable toprovide a method of immunotherapy which could be used not only toenhance, but to suppress T cell-mediated immunoresponses where suchimmunosuppression would be advantageous, for example, in transplantpatients, in patients exhibiting shock syndrome and in certain forms ofautoimmune disease.

SUMMARY OF THE INVENTION

[0009] The present invention comprises a method of immunotherapy inwhich the T cell-mediated immune response is regulated by the CD28pathway. Binding of the CD28 receptor with anti-CD28 antibodies or otherstimulatory binding equivalents induces activated T cell-mediatedlymphokine production. Immunosuppression or down-regulation is achievedby preventing CD28 receptor binding to stimulatory ligands orinactivation of the CD28 signal transduction pathway.

[0010] The method of immunotherapy of the present invention takesadvantage of the surprising and heretofore unappreciated effects ofstimulation of the CD28 surface receptor molecule of activated T cells.By activated T cells is meant cells in which the immune response hasbeen initiated or “activated,” generally but not necessarily by theinteraction of the T cell receptor (TCR)/CD3 T cell surface complex witha foreign antigen or its equivalent. While such activation results in Tcell proliferation, it results in only limited induction of T celleffector functions such as lymphokine production.

[0011] Stimulation of the CD28 cell surface molecule with anti-CD28antibody results in a marked increase of T cell lymphokine production.Surprisingly, even when the stimulation of the TCR/CD3 complex ismaximized, upon costimulation with anti-CD28, there is a substantialincrease in the levels of IL-2 lymphokine, although there is nosignificant increase in T cell proliferation over that induced byanti-TCR/CD3 alone. Even more surprisingly, not only are IL-2 levelssignificantly increased, but the levels of an entire set of lymphokines,hereinafter referred to as T_(H)CD28 lymphokines, previously notassociated with CD28 stimulation are increased. Remarkably both the Tcell proliferation and increased lymphokine production attributable toCD28 stimulation also exhibit resistance to immunosuppression bycyclosporine and glucocorticoids.

[0012] The method of immunotherapy of the present invention thusprovides a method by which the T cell-mediated immune response can beregulated by stimulating the CD28 T cell surface molecule to aid thebody in ridding itself of infection or cancer. The method of the presentinvention can also be used not only to increase T cell proliferation, ifso desired, but to augment or boost the immune response by increasingthe levels and production of an entire set of T cell lymphokines nowknown to be regulated by CD28 stimulation.

[0013] Moreover, because the effectiveness of CD28 stimulation inenhancing the T cell immune response appears to require T cellactivation or some form of stimulation of the TCR/CD3 complex, themethod of immunotherapy of the present invention can be used toselectively stimulate T cells preactivated by disease or treatment toprotect the body against a particular infection or cancer, therebyavoiding the non-specific toxicities of the methods presently used toaugment immune function. In addition, the method of immunotherapy of thepresent invention enhances T cell-mediated immune functions even underimmunosuppressed conditions, thus being of particular benefit toindividuals suffering from immunodeficiencies such as AIDS.

[0014] It will also be appreciated that although the followingdiscussion of the principles of the present invention exemplifies thepresent invention in terms of human therapy, the methods describedherein are similarly useful in veterinary applications.

[0015] A better understanding of the present invention and itsadvantages will be had from a reading of the detailed description of thepreferred embodiments taken in conjunction with the drawings andspecific examples set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a bar graph illustrating the absence of augmentation ofthe uptake of thymidine by CD28 stimulated T cells.

[0017]FIG. 2 is a bar graph illustrating the increase in uridineincorporation by CD28 stimulation of anti-CD3 stimulated T cells.

[0018]FIG. 3 is a graph illustrating the elevated cyclosporineresistance of T cell proliferation induced by CD28 stimulation.

[0019]FIG. 4 is a Northern blot analysis of the effects of cyclosporineon PMA-or anti-CD3 activated T cell lymphokine expression induced byanti-CD28.

[0020]FIG. 5 is a graph illustrating in vivo activation of T cells inmonkeys by CD28 stimulation.

[0021]FIGS. 6A and 6B are graphs representing changes in lymphocytelevels after infusion of anti-CD28 Mab.

[0022]FIGS. 7A and 7B are graphs representing in vitro production of TNFand IL-6 by PBLs under various conditions.

[0023]FIGS. 8A and 8B are graphs representing serum concentration ofIL-1 β after single and multiple doses of anti-CD28 Mab.

[0024]FIGS. 9A and 9B are graphs representing the serum concentration ofIL-6 after single and multiple doses of anti-CD28 Mab.

[0025]FIGS. 10A and 10B are graphs representing IL-6 production of invitro stimulated PBLs isolated from monkeys treated with a single bolusor multiple injections of anti-CD28 Mab.

[0026]FIG. 11 is a graph representing the inhibitory effect of CTLA-4lgon ³H-thymidine incorporation in a one-way mixed lymphocyte culture.

[0027]FIGS. 12A and 12B are photographs of cardiac allografts toillustrate histopathology.

[0028]FIG. 13 is a Kaplan-Meier life analysis of cardiac allograftsurvival after CTLA-4lg treatment.

[0029]FIG. 14 is a bar graph illustrating CTLA-4lg and cyclosporine assynergistic immunosuppressants.

[0030]FIG. 15 is a bar graph illustrating the effect of herbimycin A onCD28-stimulated IL-2 production.

[0031]FIG. 16 is a bar graph illustrating activation by SEB andanti-CD28 on purified resting T cells in the presence and absence of ablocking Mab to HLA-DR.

[0032]FIG. 17 is a bar graph illustrating activation by SEB alone or SEBand blocking Mab to HLA-DR in peripheral blood mononuclear cells.

[0033]FIG. 18 is a graph showing in vitro long term growth of CD4⁺peripheral blood T cells propagated with anti-CD3 and anti-CD28.

[0034]FIG. 19 is a Northern blot analysis of the enhancement of MRNA forIL-2 and TNF-α after costimulation with anti-CD3 and anti-CD28.

[0035]FIG. 20 is a Northern blot analysis of the ability of mitogens toinduce CTLA-4 mRNA expression.

[0036]FIG. 21 is a Northern blot analysis of the induction of CTLA-4mRNA expression by costimulation with anti-CD3 mAb and soluble anti-CD28mAb.

[0037]FIG. 22 is a graph illustrating the effects on disease progressionof CTLA-4lg treatment of syngeneic, MBP-sensitized cells used toadoptively transfer the murine autoimmune disease, ExperimentalAutoimmune Encephalomyelitis (EAE).

[0038]FIG. 23 is a graph illustrating the effect on disease progressionof CTLA-4lg or control IgG treatment of donor mice and/or isolated cellsused to adoptively transfer EAE.

[0039]FIG. 24 is a graph depicting the effect on disease severity ofdirect administration of CTLA-4lg or control human IgG to PLSJLFI/J micewith adoptively transferred EAE.

[0040]FIG. 25 is a graph illustrating the effect on disease progressionof direct administration of CTLA-4lg or control human IgG to SJL/J micewith adoptively transferred EAE.

[0041]FIG. 26 is a graph depicting the effect of direct administrationof CTLA-4lg or IgG on disease severity in SJL/J mice with adoptivelytransferred EAE.

[0042]FIG. 27 is a graph illustrating the effect on disease severity ofdirect administration of CTLA-4lg or control IgG to PLSJLFI/J micedirectly immunized with MBP and treated with PT.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] In one preferred embodiment of the immunotherapeutic method ofthe present invention, the CD28 molecule is stimulated to enhance the Tcell-mediated immune response of antigen or otherwise activated T cells.CD28 is a 44 kilodalton protein expressed on the surface of about 80%mature T cells which exhibits substantial homology to immunoglobulingenes. See Poggi, A. et al., Eur. J. Immunol., 17:1065-1068 (1987) andAruffo, A. et al., PNAS (USA), 84:8573-8577 (1987), both hereinincorporated by reference. Binding of the CD28 molecule's extracellulardomain with anti-CD28 antibodies in accordance with the method of thepresent invention results in an increase in T cell proliferation andelevated lymphokine levels.

[0044] In Specific Examples III-IV and VI-VIII, T cell activation wasaccomplished by stimulating the T cell TCR/CD3 complex (which mediatesthe specificity of the T cell immune response) with immobilized anti-CD3monoclonal antibodies, such as mAb G19-4, or by chemically stimulatingwith PMA and ionomycin. It should also be appreciated, however, thatactivation of the T cell can instead be accomplished by routes that donot directly involve CD3 stimulation, such as the stimulation of the CD2surface protein.

[0045] In practice, however, an activated T cell population will beprovided by the patient's own immune system, which, barring totalimmunosuppression, will have T cells activated in response to anyforeign or substantially elevated level of antigen present due todisease, infection, inoculation or autoimmunity. The term “foreignantigen” is used broadly herein, meaning an antigen which is either notnormally produced by the organism, or, as in carcinomas, an antigenwhich is not normally produced by the cell which is producing it. Theterm is also meant to include an antigen which should normally be seenas “self,” but, as occurs in autoimmune disease states, provokes animmune response as would a foreign antigen. By “substantially elevated”level of antigen is meant an antigen level exceeding normal ranges andhaving potentially deleterious effects to the organism due to suchelevation.

[0046] In accordance with the method of the present invention,stimulation of the CD28 molecule itself is achieved by administration ofa ligand, such as a monoclonal antibody or a portion thereof, (e.g.F(ab′)₂), having a binding specificity for and stimulatory effect onCD28. Suitable antibodies include mAb 9.3, an IgG2a antibody on depositwith the ATCC which has been widely distributed and is available (fornon-commercial purposes) upon request from Dr. Jeffrey A. Ledbetter ofOncogen Corporation, Seattle, Wash., or Mab Kolt-2. Both thesemonoclonal antibodies have been shown to have binding specificity forthe extracellular domain of CD28 as described in “Leukocyte Typing II,”Ch. 12, pgs. 147-156, ed. Reinherz, E. L. et al. (1986). Monoclonalantibody 9.3 F(ab′)₂ has also been shown to be a satisfactory ligandcapable of stimulating the CD28 receptor. It should also be understoodthat the method of the present invention contemplates the use ofchimaeric antibodies as well as non-immunoglobulin, natural andrecombinant ligands which bind the CD28 surface molecule. More recently,the natural ligand for CD28, B7/BB1, has also been identified and can beused in accordance with the principles of the present invention. SeeLinsley, P. S. et al., J. Exp. Med. 174:561 (1991). In addition, bindinghomologs of a natural ligand, whether natural or synthesized bybiochemical, immunological, recombinant or other techniques, can also beused in accordance with the principles of the present invention. It willbe appreciated that the ligands referred to herein can be utilized intheir soluble or cell-bound forms, depending on their application.Monoclonal antibody 9.3 and B7 are currently preferred stimulatoryligands.

[0047] The extracellular domain of CD28, which was sequenced by Aruffo,A. et al., PNAS (USA), 84:8573-8577 (1987), generally comprises thefollowing amino acid sequence:MetLeuArgLeuLeuLeuAlaLeuAsnLeuPheProSerIleGlnValThrGlyAsnLysIleLeuValLysGlnSerProMetLeuValAlaTyrAspAsnAlaValAsnLeuSerCysLysTyrSerTyrAsnLeuPheSerArgGluPheArgAlaSerLeuHisLysGlyLeuAspSerAlaValGluValCysValValTyrGlyAsnTyrSerGlnGlnLeuGlnValTyrSerLysThrGlyPheAsnCysAspGlyLysLeuGlyAsnGluSerValThrPheTyrLeuGlnAsnLeuTyrValAsnGlnThrAspIleTyrPheCysLysIleGluValMetTyrProProProTyrLeuAspAsnGluLysSerAsnGlyThrIleIleHisValLysGlyLysHisLeuCysProSerProLeuPheProGlyProSer LysPro

[0048] By the term “extracellular domain” as used hereinafter in thespecification and claims, is meant the amino acid sequence set forthabove, any substantial portion thereof, or any sequence havingsubstantial homology thereto.

[0049] As shown by the data of Specific Examples III-V, substantialaugmentation of the T cell-mediated immunoresponse by CD28 stimulationappears specific for activated T cells. Such specificity is ofparticular clinical importance and is one of the significant advantagesof the method of immunotherapy of the present invention. Administrationof anti-CD28 antibodies or other CD28 ligands will specifically augmentthe response of T cells which are already activated and engaged in theimmune response or those in the process of activation. It should,however, also be appreciated that CD28 stimulation may be effective evenwhere the T cells are activated after the binding of the CD28-specificligand of the present invention to CD28 receptor. Thus, the T cells ator near the tumor site or site of infection, which are being activatedby the antigens produced or present at those sites, will be selectively“boosted” by the CD28 stimulation.

[0050] Boosting of the immune response can also be beneficial to healthyindividuals, for example, in augmenting their response to antigenspresented in vaccines (see Specific Example IX). CD28 stimulationcoupled with antigen administration in accordance with the presentinvention can result in more effective immunization, not only withconventional vaccines, but in situations where an adequate immuneresponse is difficult to elicit, e.g. with human retroviruses such asHIV and some herpes viruses. Examples where CD28 stimulation of thepresent invention can be used to augment the immune response include,but are not limited to viral vaccines against measles, influenza, polio,herpes viruses (i.e. HCMV, Epstein Barr Virus, Herpes Simplex Type I and11); bacterial vaccines against whooping cough (Bordatella pertussis),tetanus (Clostridium tetanus), pneumonia (Streptococcus pneumoniae),meningitis and gonorrhea (Neisseria) and against enteropathic bacteriasuch as Salmonella, E. coli and Shigella. The principles of the presentinvention are also applicable in inoculations against parasiticinfection, including those caused by protozoal parasites, e.g. malaria,trypanosomiasis, leishmaniasis, amebiasis, toxoplasmosis, pneumocystiscarinni and babesiosis, by cestodes (e.g. tapeworm) and by otherparasitic organisms. It will also be appreciated that immunization for ahumoral response through injection of cDNA for intracellular antigenicproduction, as described in Nature 356:152 (1992), costimulated withanti-CD28 is also contemplated as within the scope of the presentinvention.

[0051] Indeed, recently CD28 engagement and signal transduction havebeen used to identify IL-13 (Minty, A. et al., Nature 362: (6417):248-50(1993)), a lymphokine involved in the inflammatory and immune response.Punnonev, J. et al., PNAS (USA) 90(8): 3730-4 (1993). When TCR/CD3stimulation is maximized, although T cell proliferation is not markedlyincreased, the levels of certain lymphokines are substantially increasedby CD28 activation, indicating an increase in cellular production ofthese lymphokines. Thus, in patients undergoing natural maximal TCR/CD3stimulation or its equivalent T cell activation in vivo due to diseaseor infection, the administration of anti-CD28 antibody or other CD28ligand to stimulate CD28 in accordance with the method of the presentinvention will result in substantially elevated lymphokine production.

[0052] The increase in lymphokine production achieved by administrationof a CD28 stimulator in accordance with the method of the presentinvention, as particularly shown in Specific Example III, surprisinglyresults in the increased production of an entire set of lymphokines,indicating that these lymphokines are under some form of CD28regulation. Part of this set of lymphokines, which includes IL-2, TNF-α,LT, IFN-γ, and IL-3 as later determined, is somewhat analogous to theT_(H)1 cell lymphokines present in the mouse which were described byMosmann, T. R. et al., Immunol. Today, 8:223-227 (1987). Although it wasoriginally believed that human IL-4 production was not increased by CD28stimulation, more recent assays as set forth in Specific Example IIIhave now also shown an increase in the production of other lymphokines,including IL-4 and IL-5 and the increased production of IL-6 and IL-1was also confirmed in Specific Example IX. It will be appreciated,however, that the term “T_(H)1 lymphokines” was originally used for easeof reference and was expressly not limited to the lymphokines listedabove, but was meant to include all lymphokines whose production isaffected or regulated by the binding or stimulation of the CD28 T cellsurface molecule. Thus the group of lymphokines affected by CD28 willhereinafter be referred to as T_(H)CD28 lymphokines, it will again beappreciated that the term T_(H)CD28 lymphokine is not intended to belimiting to the specific lymphokines listed herein. Furthermore, it willbe appreciated that the principles of the present invention can be usedin veterinary applications to increase the T cell-mediated immuneresponse and lymphokine production in animals. The term T_(H)CD28lymphokines, as used herein, is thus also meant to include analogousanimal lymphokines. Thus, by administration of anti-CD28 antibodies orother CD28 ligands in accordance with the method of the presentinvention, the production and levels of an entire set of lymphokines canbe significantly increased.

[0053] The method of immunotherapy of the present invention can also beused to facilitate the T cell-mediated immune response inimmunodepressed patients, such as those suffering from AIDS. As shown inSpecific Examples VI-VII, T cell proliferation and the increased levelsor production of CD28-regulated lymphokines continue to function even inthe presence of immunosuppression such as that caused by cyclosporine ordexamethasone. Thus administration of CD28 stimulators such as mAb 9.3or other CD28 ligands can be used to treat immunodepressed patients toincrease their in vivo lymphokine levels.

[0054] In addition, a variety of syndromes including septic shock andtumor-induced cachexia may involve activation of the CD28 pathway andaugmented production of potentially toxic levels of lymphokines. Theimmune response can also be deleterious in other situations such as inorgan transplant recipients or in autoimmune disease. Thusdown-regulation or inactivation of the CD28 pathway, as discussed morefully below and in Specific Examples X and XI, can also provideimmunotherapy for those and other clinical conditions.

[0055] It should be appreciated that administration of an anti-CD28antibody has not heretofore been seriously contemplated as a potentialimmunotherapeutic method for the substantial increase of lymphokinelevels at the sites of activated T cells. For example, the addition ofmAb 9.3 has been thought only to somewhat augment T cell proliferation,not to induce substantial increases in T_(H)CD28 lymphokine production.

[0056] Although it is not the intent herein to be bound by anyparticular mechanism by which CD28 binding regulates the T cell-mediatedimmune response, a model for the mechanism of stimulation has beenpostulated and supported with experimental data, some of which is shownin Specific Example VIII. It has previously been shown that a number oflymphokine genes undergo more rapid degradation in the cytoplasm thanmRNAs from constitutively expressed housekeeping genes, leading to thehypothesis that the instability of these inducible mRNAs has beenselected to allow for rapid regulation of gene expression. It isbelieved that the mechanism of CD28 regulation herein described andclaimed is related to the stabilization of rapidly degradable mRNAs forthe set of T_(H)CD28 lymphokines set forth above. To date, it appears noother mechanism in any eukaryotic cell system has been described todemonstrate that a cell surface activation pathway can alter geneexpression by inducing specific alteration in mRNA degradation. (A morein-depth analysis of possible models of CD28 activation is presentedlater herein.)

[0057] As shown in Specific Example IV, costimulation of CD28 andTCR/CD3 caused an increase in mRNA of the T_(H)CD28 lymphokines whichwas not the result of a generalized increase in a steady state mRNAexpression of all T cell activation-associated genes. The increase wasdisproportionate and thus could not be accounted for by the increase inpercentage of proliferating cells in culture. These data, in addition tofurther studies not detailed herein, demonstrate that activation of theCD28 surface molecule of activated T cells functions to specificallystabilize lymphokine mRNAs. Increased mRNA stability, i.e. slowerdegradation thereof, results in increased translation of the mRNA, inturn resulting in increased lymphokine production per cell. An increasein per T cell production of lymphokines that allows the T cell toinfluence the response of other inflammatory and hematopoietic cells isreferred to as paracrine production. In contrast, an increase inlymphokine levels merely due to increased cell proliferation, such asthat shown in Martin, P. J. et al., J. Immunol. 136:3282-3287 (1986), iscommonly referred to as autocrine production. For ease of reference,paracrine production is also herein referred to as “cellular” productionof lymphokines.

[0058] Thus, in accordance with the principles of the present invention,ligands with binding specificity for the CD28 molecule are administeredin a biologically compatible form suitable for administration in vivo tostimulate the CD28 pathway. By “stimulation of the CD28 pathway” ismeant the stimulation of the CD28 molecule resulting in increased T cellproduction of T_(H)CD28 lymphokines. By “biologically compatible formsuitable for administration in vivo” is meant a form of the ligand to beadministered in which the toxic effects, if any, are outweighed by thetherapeutic effects of the ligand. Administration of the CD28 ligand canbe in any suitable pharmacological form, including but not limited tointravenous injection of the ligand in solution.

[0059] It should be understood that, although the models for CD28regulation of lymphokine production are described with respect tostimulation and enhancement of lymphokine levels, as noted above,down-regulation or inhibition of the CD28 pathway is also in accordancewith the principles of the present invention. Down-regulation orsuppression of the immune response is of particular clinical interestfor a variety of conditions, including septic shock, tumor-inducedcachexia, autoimmune diseases and for patients receiving heart, lung,kidney, pancreas, liver and other organ transplants. One preferredapproach to down-regulation is the blocking of the CD28 receptorstimulatory binding site on its natural ligand. For example, CTLA-4(discussed in more detail below), which shares 32% amino acid homologywith CD28 and appears to have greater binding affinity for B7 than CD28,can be used to bind B7 and prevent CD28 binding and activation thereby.See Linsley, P. S. et al., J. Exp. Med., 174:561 (1991). Such regulationhas been accomplished in vivo as described in Specific Example X. Inthis Example, acute rejection of fully MHC-mismatched cardiac allograftswas prevented by blocking B7-dependent T cell activation, i.e. CD28binding, with the soluble recombinant fusion protein CTLA-4lg. Theimmunosuppression observed with CTLA-4lg did not result in significantalterations in circulating T cell subsets. It will be appreciated thatother B7-binding ligands such as a monoclonal antibody to B7 can besimilarly employed. In addition, CTLA-4lg treatment can be efficaciousin the treatment of autoimmune diseases, as shown in the murine modelfor multiple sclerosis (i.e. Experimental Autoimmune Encephalomyelitis(EAE)). CTLA-4lg treatment of T cells isolated from a mouse immunizedwith Myelin Basic Protein (MBP) results in reduced disease severity whenthe treated cells are introduced into a syngeneic animal. Likewise, whenmice immunized with MBP and injected with pertussis toxin (PT) aretreated directly with CTLA-4lg, disease severity is reduced. Thesefindings confirm the in vivo immunosuppressive effects of CTLA-4lgtreatment. Thus, administration of CTLA-4 can provide an effectivetherapeutic strategy for the treatment of autoimmune diseases. Thoseskilled in the art will also appreciate that the cell lines and animalmodels used to exemplify the present invention are recognized predictorsof efficacy in humans.

[0060] It will be appreciated that down-regulation can also beaccomplished by blocking CD28 receptor binding to B7 by occupying theCD28 binding site with nonstimulatory ligands which may mimicstimulatory ligands but do not result in activation of the CD28 pathway,e.g. Fabs, modified natural, synthetic, recombinant or other ligandswhich do not crosslink or otherwise do not activate receptors.

[0061] As discussed above and shown in the Specific Examples, theblockade of stimulatory ligands which bind to CD28 and activate the CD28pathway (e.g. B7) or the blocking of the CD28 binding site can reducethe increased lymphokine expression which occurs upon CD28 activation.Thus, just as manipulation of the CD28 pathway can be used to enhance Tcell immune responses, it can also be used to suppress such responses.Since unregulated lymphokine production has been implicated in theaetiology of autoimmunity, CD28-mediated immunosuppression can beexploited to treat various autoimmune diseases. Methods of suppressingthe CD28 pathway in accordance with the present invention are desirablesince this pathway is resistant to the effects of cyclosporine, which iscommonly used as an immunosuppressive agent in the treatment ofautoimmune diseases. Immunosuppression via the CD28 pathway can restoreimmunoregulation and thus reduce the pathologic effects of suchautoimmune diseases as systemic lupus erythematosis, rheumatoidarthritis, hemolytic anemia, myasthenia gravis, schleroderma, Sjögren'ssyndrome, ulcerative colitis, multiple sclerosis, and a host of othersystemic as well as organ-specific autoimmune diseases.

[0062] Administration of stimulatory ligands (e.g. mAb 9.3, Kolt, B7) orinhibitory ligands (e.g. CTLA-4lg, Fab fragments of mAb 9.3, and thelike) can be in any suitable pharmacological form, includingparenterally or topically. Pharmaceutical compositions may also take theform of ointments, gels, pastes, creams, sprays, lotions, suspensions,solutions and emulsions of the active ingredient in aqueous ornonaqueous diluents, syrups, granulates or powders. In addition to aligand of the present invention, the pharmaceutical compositions canalso contain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

[0063] More particularly, a ligand of the present invention (alsoreferred to herein as the active ingredient) may be administered by anysuitable route, including parenteral (including subcutaneous,intramuscular, intravenous and intradermal), topical (includingtransdermal, buccal and sublingual), rectal, vaginal, nasal andpulmonary. Although the preferred route of administration is currentlyparenteral, it will be appreciated that the delivery route of choicewill vary with the condition and age of the recipient and the nature ofthe disease or condition being treated.

[0064] While it is possible for the active ingredient to be administeredalone, it is preferable to present it as a pharmaceutical formulationcomprising at least one active ingredient, as defined above, togetherwith one or more pharmaceutically acceptable carriers therefor andoptionally other therapeutic agents. Each carrier must be “acceptable”in the sense of being compatible with other ingredients of theformulation and not injurious to the patient.

[0065] Formulations suitable for parenteral administration includeaqueous and non-aqueous isotonic sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations may be presented in unit-dose ormulti-dose sealed containers, for example, ampoules and vials, and maybe stored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared form sterile powders, granules andtablets of the kind previously described.

[0066] Formulations include those suitable for parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal), topical(including transdermal, buccal and sublingual), rectal, vaginal, nasaland pulmonary administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. Such methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociated the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.

[0067] Pharmaceutical compositions for topical administration accordingto the present invention may be formulated as an ointment, cream,suspension, lotion, solution, paste, gel, spray aerosol or oil.Alternatively, a formulation may comprise a patch or a dressing such asa bandage or adhesive plaster impregnated with active ingredients andoptionally one or more excipients or diluents. Formulations suitable fortopical administration in the mouth include mouthwashes comprising theactive ingredient in a suitable liquid carrier. It will also beappreciated that in a carrier suitable to preserve efficacy of theligand, oral administration is also contemplated.

[0068] For conditions of the eye or other external tissues, e.g. mouthand skin, the formulations are preferably applied as a topical ointmentor cream containing the active ingredient. When formulated in anointment, the active ingredient may be employed with either a paraffinicor a water-miscible ointment base. Alternatively, the active ingredientsmay be formulated in a cream with an oil-in-water cream base. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active ingredient through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethylsulphoxide and related analogues. Formulations suitablefor topical administration to the eye also include eye drops wherein theactive ingredient is dissolved or suspended in a suitable carrier,especially an aqueous solvent for the active ingredient.

[0069] Formulations for rectal administration may be presented as asuppository with a suitable base. Formulations for vaginaladministration may be presented as pessaries, tampons, creams, gels,pastes, foams or spray formulations containing in addition to the activeingredient, such carriers as are known in the art to be appropriate.Formulations suitable for nasal administration, include formulationswherein the carrier is a liquid for administrations, for example, anasal spray or a nasal drops, include aqueous or oily solutions of theactive ingredient.

[0070] Preferred unit dosage formulations are those containing a dailydose or unit, daily subdose, or an appropriate fraction thereof, of theactive ingredient. In any event, in practicing the present invention,the amount of active ingredient to be used or administered, alone or incombination with other agents, will vary with the patient being treatedand will be monitored on a patient-by-patient basis by the physician.Generally, a therapeutically effective amount of the vaccine will beadministered for a therapeutically effective duration. By“therapeutically effective amount” and “therapeutically effectiveduration” are meant an amount and duration to achieve the result desiredin accordance with the present invention without undue adverse or withmedically acceptable physiological effects, which effects can bedetermined by those skilled in the medical arts. It will also beappreciated that, particularly when a natural ligand is used in thepractice of the invention, its isolation or production should render itsubstantially free of undesirable contaminants such as other proteins(i.e. “substantially pure”) which may adversely impact on its efficacyor use. Acceptable levels of purity can be determined by those skilledin the pharmaceutical and medical arts and can depend on the specificligand and composition and its intended use.

[0071] It should also be understood that in addition to the ingredientsparticularly mentioned above, the formulations of this invention mayinclude other agents conventional in the art having regard to the typeof formulation in question. In accordance with the present invention,ligands may also be presented for the use in the form of veterinaryformulations, which may be prepared, by methods conventional in the art.

[0072] Accumulating evidence suggests that in addition to T cellreceptor occupancy, other costimulatory signals are required to induce acomplete T cell-mediated immune response. The CD28 receptor expressed onT cells serves as a surface component of a novel signal transductionpathway that can induce paracrine levels of cellular production oflymphokines. Interaction of CD28 with its natural ligand B7 which isexpressed on the surface of activated B cells macrophages or dendriticcells can act as a costimulus to induce high level lymphokine productionin antigen receptor-activated T cells. Thus, another approach todown-regulation is to inhibit the activation of the CD28 signaltransduction pathway as described below.

[0073] Although the CD28 signal transduction pathway is not entirelyunderstood, Specific Example XI demonstrates that binding of CD28induces protein tyrosine phosphorylation distinct from T cell receptor(TCR)-induced tyrosine phosphorylation. For example, TCR-inducedtyrosine phosphorylation occurs in both resting and activated T cells,while CD28-induced tyrosine phosphorylation occurs primarily inpreviously activated T cells. Most striking were the results after CD28receptor ligation by cell-bound B7, where phosphorylation wasconsistently detectable on only a single substrate. Experiments usingthe Jurkat E6-1 T cell line indicated an absolute requirement for PMApretreatment in order to observe CD28-induced tyrosine phosphorylation.In contrast, there was no requirement for cellular preactivation in theJurkat J32 line, while, as noted above, there was a relative requirementfor PMA or TCR prestimulation of normal T cells in order to induce CD28responsiveness. Studies with Jurkat mutants further indicated thatCD28-induced tyrosine phosphorylation and biologic function can occur inthe absence of the TCR. In this respect, CD28 appears to be unique inthat other accessory molecules involved in T cell activation, such asCD2, Ly-6, Thy-1, and CD5 appear to require the presence of the TCR.Thus, specific tyrosine phosphorylation appears to occur directly as aresult of CD28 ligand binding and is involved in transducing the signaldelivered through CD28 by accessory cells that express the B7/BB1receptor.

[0074] Studies with an inhibitor of the src family of tyrosine kinases,herbimycin A, and with tyrosine phosphatase, as described in SpecificExample XI, further show that the functional effects of CD28 stimulationon lymphokine gene expression require the above-described proteintyrosine phosphorylation. The data on tyrosine phosphorylationinhibitors thus demonstrate that inactivation of CD28-mediated signaltransduction can also be used to down-regulate lymphokine production inaccordance with the principles of the present invention.

[0075] Immunotherapy through CD28 stimulation in accordance with thepresent invention also has clinical applicability in the treatment ofbone marrow transplant recipients. The success of autologous andallogeneic bone marrow transplantation (BMT) has generally been limitedby recurrent malignancy, graft-vs-host disease (GVHD), and thelife-threatening immune deficiency that occurs after BMT. One approachto overcoming these problems has been the adoptive transfer oflymphocytes in combination with lymphokine infusions, to accelerateimmune reconstitution or mediate cytotoxicity directed at malignantcells. The side effects attending such transfer with lymphokineinfusions are, however, quite significant.

[0076] Thus, T cell proliferation and lymphokine synthesis in theabsence of exogenously added IL-2 in response to CD3 and CD28costimulation, as described in Specific Example XIII, provides a uniqueopportunity for clinical use of adoptive transfer of activated T cellsto repair T cell defect in vivo without exogenous lymphokine infusions.The studies detailed in Specific Example XIV show that defective invitro proliferative responses to anti-CD3 (OKT3 or G19-4) can berepaired by adding mAb 9.3 to the cultures. See Joshi, I. et al., BloodSuppl. (Abstract) (1991). Costimulation of T cells with OKT3/9.3repaired proliferative responses as a result of increasing the levels ofMRNA expression for cytokines/lymphokines such as IL-2, GM-CSF, andTNFα. See Joshi, supra; Perrin, P. J. et al, Blood Suppl. (Abstract)(1991). Purified normal CD4⁺ cells can thus be costimulated withOKT3/9.3 to expand and secrete lymphokines for long periods of time.Preclinical studies using mAb 9.3 stimulation have shown no untowardeffects in monkeys. Although CD3-stimulated cells (CTC) provide helperfactors to normal B cells, CD3-CD28 costimulated cells appear even moreeffective in producing helper factors than CD3-stimulated T cells. Thus,costimulation may also enhance the growth of helper cells and cytotoxicT cells for adoptive immunotherapy after BMT. The administration in vivoof T cells that have been expanded in vitro, will provide two prominentbenefits in marrow transplantation. The ability of CD28-treated cells toproduce many lymphokines which have a strong positive effect onhematopoiesis, such as GM-CSF, IL-3, and IL-6, should accelerateengraftment after marrow transplantation. In addition, the ability ofanti-CD28 to trigger cytotoxicity and to cause the production oflymphokines such as TNF is a novel form of adoptive immunotherapy thatshould augment the anti-neoplastic efficacy of bone marrowtransplantation.

[0077] The possible role of CD28 in anergy was also examined. Generally,the activation of a quiescent T cell is initiated through stimulation ofthe T cell antigen receptor. This activation can occur either throughengagement of an antigenic peptide presented in the antigen bindinggroove of a self-encoded MHC molecule or by engagement of a foreign MHCmolecule. However, while this receptor-mediated activation event isrequired for the initiation of a T cell response in a quiescent cell,recent studies have demonstrated that signals transduced by the antigenreceptor alone are not sufficient to lead to an effective Tcell-mediated immune response. Several in vitro models suggest that, infact, T cell receptor (TCR)/CD3 activation alone of a quiescent T cellleads to the induction of a state in which the T cell becomes anergic tofurther stimulations through its antigen-specific receptor. This stateis relatively long-lived and, for at least several weeks, renders thatcell incapable of further response upon antigenic stimulation. It ishypothesized that this isolated activation of the TCR/CD3 complex alonein the absence of additional T cell costimulatory molecules plays animportant role in regulating a peripheral immune response by preventingT cells from responding to self antigens in the periphery.

[0078] Thus, for T cells to mount a proliferative response and initiatea cell-mediated immune response, a quiescent T cell normally requiresstimulation not only through its antigen-specific T cell receptor butalso through a second receptor which provides additional costimulatorysignals to the cell. The data set forth herein, e.g. in Specific ExampleXII, demonstrates that CD28 provides an essential costimulatory signalfor T cell responses in vitro and in vivo. Thus, the CD28 receptor'sability to augment T cell lymphokine production not only results in theinitiation of a cell-mediated immune response, but also prevents theinduction of anergy in a quiescent T cell.

[0079] The role of CD28 in the prevention of programmed cell death hasalso been tested. The induction of cell death has a major role in theelimination of self-reactive or non-reactive T cells in the thymus. Inthe thymus, it is thought that T cell receptor signals are able toinduce programmed cell death, in a selective and specific fashioninvolving cells that express T cell receptors specific forself-antigens. As described in Turka, L. A. et al., J. Immunol.,144:1646-53 (1990), CD28 is expressed in developing T cells in thethymus, and the binding of mAb 9.3 prevents thymocyte cell death.Programmed cell death is also thought to occur in mature T cells inperipheral lymphoid organs. Signals delivered through the T cellreceptor can induce cell death (see Newell, M. K., et al., Nature,347:286-8 (1990)). It has also been proposed that cell death may have arole in certain forms of immunopathology. For example, in HIV-1infection it has been proposed that the progressive immunodeficiency maybe the result of immunologically-mediated cell death, rather than adirect consequence of viral-induced cytopathic effects. See Ameisen, J.C. et al., Immunol. Today, 4:102 (1991); also see Groux H., et al., J.Exp. Med. 175:331-340 (1992). The results in Specific Example XIII showthat CD28 can prevent cell death in mature T cells. Thus, abnormalexpression or activation of CD28 may have a role in immunopathology ofcertain autoimmune disorders such as systemic lupus erythematosus, adisorder characterized by abnormally self-reactive T cells that havefailed to undergo elimination in the thymus or escape from anergicstates in the peripheral lymphoid system. Similarly, the ability toinduce CD28 activation may be beneficial in disorders characterized byprogressive T cell depletion such as HIV-1 infection.

[0080] It was also demonstrated in Specific Example XII thatsuperantigens SEA and SEB, which do not require traditional processingfor binding to MHC, can directly activate purified T cells in theabsence of accessory cells as determined by a transition from G₀ to G₁and induction of IL-2 receptor expression. However, neither SEA nor SEBalone was sufficient to result in T cell proliferation. The induction ofT cell proliferation by SEB or SEA required the addition of a secondcostimulatory signal. This could be provided by either accessory cellsor monoclonal antibody stimulation of CD28. As previously reported, Tcell proliferation induced by enterotoxin in the presence of accessorycells was partially inhibited by a blocking antibody (HLA-DR) againstclass II MHC. In contrast, in purified T cells when costimulation wasprovided through CD28, proliferation was not inhibited by class IIantibody and HLA-DR expression was not detectable. In addition,costimulation through CD28 was partially resistant to the effects ofcyclosporine. These results demonstrate that CD28 costimulation issufficient to induce lymphokine production and subsequent proliferationof enterotoxin-activated T cells, and that this effect is independent ofclass II MHC expression. This prevention of in vivo CD28 activation ofsuperantigen-activated cells such as those occurring in toxic shocksyndrome and rheumatoid or lyme arthritis, may substantially decreasedisease morbidity.

[0081] Although as noted previously the present invention is notintended to be restricted to specific mechanisms of activation, the roleof CTLA-4 in the CD28 activation pathway has been examined and modelsconsistent with the data presented herein have been postulated. (Seee.g. Specific Example XV.) Recent work in our laboratory has shown thatthe CTLA-4 gene lies immediately adjacent to CD28 on chromosome 2, witha similar genomic organization and 32% amino acid homology. Based ontheir chromosomal localization and sequence and organizationalsimilarities, CD28 and CTLA-4 likely represent evolutionary geneduplication. By standard nomenclature they might thus more appropriatelybe named CD28α and CD28β, although the terms CD28 and CTLA-4 areretained herein.

[0082] Although CTLA-4 is not expressed on quiescent lymphoid cells, itsexpression at the RNA level can be rapidly induced upon T cellactivation. Two potential mechanisms by which CTLA-4 might function arepostulated as follows. First, since CD28 and CTLA-4 contain an unpairedcysteine in their extracellular domain, this cysteine residue may beused to form crosslinked dimeric receptors on the surface. If this werethe case, it may suggest that CTLA-4 is normally expressed on thesurface as a heterodimer with CD28. Under such conditions, the higheraffinity of CTLA-4 for the natural ligand B7 might in the dimeric statelead to a higher affinity receptor with enhanced signaling capabilities.This might allow for an enhanced signal transduction capability throughthe CD28-CTLA-4 heterodimer in an antigen-activated cell. In addition,if CD28 and CTLA-4 are found primarily in activated cells in aheterodimeric state, this might account for observations thatCD28-containing receptors have enhanced signaling capabilities inactivated cells.

[0083] On the other hand, the data presented herein are also compatiblewith a model in which CTLA-4 is induced upon T cell activation as acompetitive inhibitor of CD28 and is used to down-modulate an ongoingimmune response by inhibiting further interactions between B7 and CD28on the surface. In addition, it is quite possible that the CTLA-4expressed on the surface is also expressed in a shed form, and this shedform of the receptor acts as a soluble competitive inhibitor of anongoing B7-CD28 interaction, thereby preventing the antigen-presentingcell from activating additional T cells in its environment. Thus, theability of T cells to produce an additional isoform of CD28, i.e.CTLA-4, suggests that the interplay of expression of CD28 and CTLA-4 hasprofound effects on the ability of T cells to be activated through aCD28-containing receptor.

[0084] CD28 pathway activation and inhibition studies indicate that theability of the CD28 natural ligand B7 to activate a T cell to augmentlymphokine production is entirely mediated through a CD28-containingreceptor, either a CD28 homodimer or a CD28-CTLA-4 heterodimer. Thus,the data suggest that a CTLA-4 homodimer is not critical in T cellactivation, but may play an important role in down-modulation of T celllymphokine production, while a CD28-CTLA-4 heterodimer may account forthe enhanced signaling properties of CD28-containing receptors upon Tcell activation.

[0085] The role of CTLA-4 in CD28-mediated signal transduction event mayexplain why the novel and profound effects of CD28 on normal T cellactivation encountered and described herein were not previously observedin human T cell lines. Earlier work on the CD28 pathway occurred in celllines such as Jurkat human T cells. Extensive attempts to stimulatethese cells to express CTLA-4 have been entirely negative (see FIG. 20).In contrast, standard activation events that lead to cell cycleprogression of normal T cells either through chemical mitogens such asphytohemagglutinin (PHA) and phorbol myristate acetate (PMA) leads torapid induction of CTLA-4 expression as does crosslinking of the TCR/CD3complex. Therefore, the inability of previous investigators toappreciate or harness the CD28 activation pathway to enhance cellularproduction of lymphokines was likely due to the lack of expression ofthe CTLA-4 isoform of CD28 in these cell lines. Interestingly,costimulation of resting T cells with anti-CD28 monoclonal antibodiesenhances the expression of the CTLA-4 gene (see FIG. 21). Thus, the CD28activation pathway in normal cells may in fact involve a positivefeedback loop in which initial CD28 stimulation through the CD28homodimer enhances the expression of CTLA-4 thus leading to enhancedheterodimer expression and signal transduction. Alternatively, theenhanced CTLA-4 may lead to the production of a receptor which competesfor CD28 signal transduction thus leading to the ultimate termination oflymphokine production and acts as a negative feedback loop to downmodulate an ongoing CD28-mediated lymphokine production.

SPECIFIC EXAMPLE I Preparation of CD28 Stimulator Monoclonal Antibody9.3

[0086] The monoclonal antibody (mAb) 9.3, an IgG2a monoclonal antibodywhich binds to the extracellular domain of the CD28 molecule, wasproduced by a hybrid cell line originally derived as described by Hansenet al., Immunogen., 10:247-260 (1980). Ascites fluid containing hightiter monoclonal antibody 9.3 was prepared by intraperitonealinoculation of 5-10×10⁶ hybrid cells into a Balb/C×C57BL/6 F₁ mice whichhad been primed intraperitoneally with 0.5 ml of Pristane (AldrichChemical Co., Milwaukee, Wis.). The monoclonal antibody 9.3 was purifiedfrom ascites fluid on a staphylococcal protein-A sepharose column asdescribed by Hardy, R., “Handbook of Experimental Immunology,” Ch. 13(1986).

[0087] Prior to use in functional assays, purified mAb 9.3 was dialyzedextensively against phosphate buffered saline (KCl 0.2 grams/liter dH₂O;KH₂PO₄ 0.2 grams/liter dH₂O; NaCl 8.0 grams/liter dH2O; Na₂HPO₄.7H₂O2.16 grams/liter dH₂O) and then filtered through a 0.22 cubic micronsterile filter (Acrodisc, Gelman Sciences, Ann Arbor, Mich.). The mAb9.3 preparation was cleared of aggregates by centrifugation at 100,000×g for 45 m at 20° C. The resulting purified mAb 9.3 was resuspended inphosphate buffered saline to a final concentration of 200 μg/ml asdetermined by OD₂₈₀ analysis and stored at 4° C. prior to use.

SPECIFIC EXAMPLE II Isolation of CD28⁺ T Cells

[0088] Buffy coats were obtained by leukopheresis of healthy donors 21to 31 years of age. Peripheral blood lymphocytes (PBL), approximately2.5×10⁹, were isolated from the buffy coat by Lymphocyte SeparationMedium (Litton Bionetics, Kensington, Md.) density gradientcentrifugation. The CD28⁺ subset of T cells was then isolated from thePBL by negative selection using immunoabsorption, taking advantage ofthe reciprocal and non-overlapping distribution of the CD11 and CD28surface antigens as described by Yamada et al., Eur. J. Immunol.,15:1164-1688 (1985). PBL were suspended at approximately 20×10⁶/ml inRPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) containing 20mM HEPES buffer (pH 7.4) (GIBCO Laboratories, Grand Island, N.Y.), 5 mMEDTA (SIGMA Chemical Co., St. Louis, Mo.) and 5% heat-activated human ABserum (Pel-Freez, Brown Deer, Wis.). The cells were incubated at 4° C.on a rotator with saturating amounts of monoclonal antibodies 60.1(anti-CD11 a) (see Bernstein, I. D. et al., “Leukocyte Typing II,” Vol.3, pgs. 1-25, ed. Reinherz, E. L. et al., (1986); 1F5 (anti-CD20) (seeClark, E. A. et al., PNAS(USA), 82:1766-1770 (1985)); FC-2 (anti-CD16)(see June, C. H. et al., J. Clin. Invest., 77: 1224-1232 (1986)); andanti-CD14 for 20 m. This mixture of antibodies coated all B cells,monocytes, large granular lymphocytes and CD28⁻ T cells with mouseimmunoglobulin. The cells were washed three times with PBS to removeunbound antibody, and then incubated for 1 h at 4° C. with goatanti-mouse immunoglobulin-coated magnetic particles (Dynal, Inc., FortLee, N.J.) at a ratio of 3 magnetic particles per cell. Antibody-coatedcells that were bound to magnetic particles were then removed bymagnetic separation as described by Lea, T. et al., Scan. J. Immunol.,22:207-216 (1985). Typically, approximately 700×10⁶ CD28⁺ T cells wererecovered. Cell purification was routinely monitored by flow cytometryand histochemistry. Flow cytometry was performed as described byLedbetter, J. A. et al., “Lymphocyte Surface Antigens,” pgs. 119-129(ed. Heise, E., 1984). Briefly, CD28⁺ T cells were stained withfluorescein isothiocyanate (FITC)-conjugated anti-CD2 mAb OKT11(Coulter, Hialeah, Fla.) and with FITC-conjugated anti-CD28 mAb 9.3 asdescribed by Goding, J. W., “Monoclonal Antibodies Principles andPractice,” p. 230 (ed. Goding, J. W., 1983). CD28⁺ T cells were over 99%positive with FITC-conjugated monoclonal antibody OKT11 and over 98%positive FITC-conjugated monoclonal antibody 9.3 when compared to anon-binding, isotype-matched, FITC-labeled control antibody (Coulter,Hialeah, Fla.). Residual monocytes were quantitated by staining fornon-specific esterase using a commercially available kit obtained fromSigma Chemical Co., St. Louis, Mo., and were less than 0.1% in all cellpopulations used in this study. Viability was approximately 98% asmeasured by trypan blue exclusion as described by Mishell, B. B. et al.,“Selected Methods in Cell. Immunology,” pgs.16-17 (1980).

SPECIFIC EXAMPLE III Increased Cellular Production of Human T_(H)CD28Lymph kines by CD28 Stimulation by Monoclonal Antibody 9.3

[0089] A. Increased Production of IL-2, TNF-α, IFN-γ and GM-CSF.

[0090] CD28⁺ T cells were cultured at approximately 1×10⁵ cells/well inthe presence of various combinations of stimulators. The stimulatorsincluded phorbol myristate acetate (PMA) (LC Services Corporation,Woburn, Mass.) at 3 ng/ml conc.; anti-CD28 mAb 9.3 at 100 ng/ml;anti-CD3 mAb G19-4 at 200 ng/ml which was immobilized by adsorbing tothe surface of plastic tissue culture plates as previously described byGeppert, et al., J. Immunol., 138:1660-1666 (1987); also Ledbetter, etal., J. Immunol., 135: 2331-2336 (1985); ionomycin (lono) (Calbiochem.,San Diego, Calif.) at 100 ng/ml. Culture supernatants were harvested at24 h and serial dilutions assayed for the presence of T_(H)CD28lymphokines.

[0091] Specifically, IL-2 was assayed using a bioassay as previouslydescribed by Gillis et al., Nature, 268:154-156 (1977). One unit (U) wasdefined as the amount of IL-2 needed to induce half maximalproliferation of 7×10³ CTLL-2 (a human cytotoxic T cell line) cells at24 h of culture. In separate experiments, the relative levels of IL-2for each of the culture conditions above were independently confirmedusing a commercially available ELISA assay (Genzyme Corp., Boston,Mass.). TNF-α/LT levels were measured using a semi-automated L929fibroblast lytic assay as previously described by Kunkel et al., J.Biol. Chem., 263:5380-5384 (1988). Units of TNF-α/LT were defined usingan internal standard for TNF-α (Genzyme Corp., Boston Mass.). Theindependent presence of both TNF-α and LT was confirmed by the abilityof a monoclonal antibody specific for each cytokine to partially inhibitcell lysis mediated by the supernatant from cells costimulated withimmobilized anti-CD3 mAb G19-4 and anti-CD28 mAb 9.3. IFN-γ was measuredby radioimmunoassay using a commercially available kit (Centocor,Malvern, Pa.). Units for IFN-γ were determined from a standard curveusing ¹²⁵I-labeled human IFN-γ provided in the test kit. GM-CSF wasdetected by stimulation of proliferation of the human GM-CSF-dependentcell line AML-193, as described by Lange et al., Blood, 70:192-199(1987), in the presence of neutralizing monoclonal antibodies to TNF-αand LT. The ³H-thymidine uptake induced by 10 ng/ml of purified GM-CSF(Genetics Institute, Cambridge, Mass.) was defined as 100 U. Separatealiquots of cells were recovered 48 h after stimulation and assayed forthe percentage of cells in late stages of the cell cycle (S+G₂+M) bystaining of cells with propidium iodide and analysis by flow cytometryas previously described by Thompson et al., Nature, 314:363-366 (1985).

[0092] As shown in Table 1, CD28 stimulation of CD3-stimulated T cellsresulted in marked increases in cellular production of IL-2, TNF-α,IFN-γ and GM-CSF. TABLE 1 Increased Cellular Production of T_(H)CD28Lymphokines by CD28 Stimulation IL-2 TNF-α/LT IFN-γ GM-CSF S + G₂ + MSTIMULUS (U/ml) (U/ml) (U/ml)  (U/ml) (%) Medium <2 0 0   0 4.6 PMA <2 00 NT 5.5 anti-CD28 <2 5 0   0 6.5 anti-CD28 + 435 300 24  150 48.9 PMAanti-CD3^(i) 36 50 24  120 39.7 anti-CD3^(i) + 1200 400 74 1050 44.7anti-CD28 lonomycin <2 0 0 NT 6.6 lonomycin + 200 5 37 NT 43.6 PMAlonomycin + 1640 320 128 NT 43.5 PMA + anti- CD28

[0093] B. Effects of Anti-CD28 Stimulation on T cell IL-4, IL-5 and IL-3Secretion.

[0094] Previous studies of the effects of anti-CD28 Mab stimulation on Tcell production of lymphokines of the T_(H)2 type were limited to thefirst few days of stimulation. In those studies IL-4 could not bedetected after anti-CD28 stimulation (noted in Thompson et al., PNAS86:1333 (1989)). On reexamination of this question, it was found thatanti-CD28 can augment production of IL-4 and of IL-5, and thus, augmentsproduction of T_(H)2 lymphokines as well as the T_(H)1 type lymphokinespreviously described. As can be seen in Table 2, small amounts of bothIL-4 and IL-5 can be detected after 24 h of stimulation with anti-CD3plus anti-CD28. However, when cells are restimulated after 8 days inculture, large amounts of both IL-4 and IL-5 are secreted. As shown inTable 3, similar results were found when the CD28 subset of T cells werestimulated with the combination of phorbol myristate acetate (PMA) andanti-CD28 mAb. These results indicated that small amounts of IL-4 andIL-5 can be detected after initial stimulation of resting T cells withanti-CD28. However, with continued stimulation, differentiation occurs,and large amounts of IL-4 and, particularly, of IL-5 are produced, whilelesser amounts of IL-2 and γ-IFN are also produced. TABLE 2 Effects ofanti-CD3 and anti-CD28 Treatment f IL-4 and IL-5 Production by T cellsINITIAL RESTIMULATION IL-4 IL-5 IFN IL-2 IL-4 IL-5 IFN IL-2 STIMULUSpg/ml pg/ml pg/ml U/ml pg/ml pg/ml pg/ml U/ml Medium <1 <1 <1 <0.1 <1 <1<1 <0.1 anti-CD3 <1 <1 630 2.3 220 225 246 0.02 anti-CD3 + 207 137 8877.0 498 2545 379 0.2 anti-CD28

[0095] The data in Table 2 above were obtained using the followingprotocol: CD28⁺ T cells were isolated by negative selection usingmonoclonal antibodies and magnetic immunobeads as described in SpecificExample II. The cells were cultured at 1×10⁶/ml in RPMI mediumcontaining 10% FCS (Medium), or in culture wells containing anti-CD3monoclonal antibody G19-4 absorbed to the plastic, or plastic adsorbedanti-CD3 plus anti-CD28 mAb 9.3 added in solution at 0.5 μg/ml.Supernatants from the cell culture were analyzed for lymphokineconcentration using commercially available ELISA kits and the valuesexpressed as pg/ml for IL-4, IL-5 and γ-IFN, or as units per ml, forIL-2. Supernatants were analyzed after 24 h of culture (Initial) oralternatively, the cells were cultured for 8 days in the above forms ofstimulation, the cells were recovered, washed, and then restimulatedwith their original treatment, and the supernatants analyzed after afurther 24 h of stimulation (Restimulation). The values represent meansof duplicate cultures. TABLE 3 Effects of PMA and anti-CD28 Treatment onIL-4 and IL-5 Production by T cells INITIAL RESTIMULATION IL-4 IL-5 IFNIL-2 IL-4 IL-5 IFN IL-2 STIMULUS pg/ml  pg/ml pg/ml U/ml pg/ml pg/mlpg/ml U/ml Medium <1 <1 <1 <0.1 <1 <1 <1 <0.1 PMA <1 <1 152 .07 <1 <1 <10.6 anti-CD28 + <1 187 558 8.1 285 392 335 10.3 PMA

[0096] The data in Table 3 above were obtained using the followingprotocol: CD28⁺ T cells were isolated by negative selection usingmonoclonal antibodies and magnetic immunobeads as described in SpecificExample II. The cells were cultured at 1×10⁶/ml in RPMI mediumcontaining 10% FSC (Medium), or in medium plus PMA 3 ng/ml, or in PMAplus anti-CD28 Mab 9.3 at 0.5 μg/ml. Supernatants from the cell culturewere analyzed for lymphokine concentration using commercially availableELISA kits and the values expressed as pg/ml for IL-4, IL-5 and γ-IFN.Supernatants were analyzed after 24 h of culture (Initial) or,alternatively, the cells were cultured for 8 days in the above forms ofstimulation, and then restimulated, and the supernatants analyzed aftera further 24 h period of stimulation (Restimulation). The valuesrepresent means of duplicate cultures.

[0097] Induction of IL-3 expression in T cells after anti-CD28treatment. IL-3 is a multilineage hematopoietic growth factor that isprimarily produced by T cells, and is generally considered to beproduced by T_(H)1 cells. The experimental protocol and the findingsdescribed herein are described in detail in Guba, S. C. et al., J Clin.Invest. 84(6):1701-1706 (1989), incorporated herein by reference.

[0098] PBL were isolated as described previously. The CD28⁺ subset of Tcells was isolated by negative selection as described by June, C. H. etal., Mol. Cell. Biol. 7:4472-4481 (1987). In some experiments, the CD28⁺subset of T cells was isolated by incubating PBL with mAB 9.3, and thenremoving the CD28⁺ cells with goat anti-mouse coated magnetic beads(Advanced Magnetics Institute, Cambridge, Mass.). Northern (RNA) blotanalysis was done as described by June, C. H. et al., Mol. Cell. Biol.7:4472-4481 (1987). The IL-3 probe was a 1.0 kb Xho I cDNA fragment.

[0099] To determine if stimulation of the TCR/CD3 pathway of T cellactivation induced IL-3 gene expression, CD28⁺ T cells were stimulatedwith maximal amounts of plastic immobilized anti-CD3 mAb in the presenceor absence of 9.3 mAb 1 μg/ml for 1 to 36 h. As shown in Table 4,anti-CD28 resulted in a 3 to 5-fold augmentation of IL-3 mRNA expressionover that induced by anti-CD3 alone. CD28 did not change the kinetics ofIL-3 gene expression, which was at peak levels at 6 h after anti-CD3 orafter anti-CD3+anti-CD28 treatment. Further experiments showed that IL-3gene expression was restricted to the CD28⁺ subset of T cells, asdetermined by Northern analysis (Table 4). The stability of IL-3 mRNAwas also determined. T cells were treated for 3 h with anti-CD3 oranti-CD3 plus anti-CD28 mAb to induce IL-3 mRNA expression. At 3 h,actinomycin D was added to the culture to inhibit further RNA synthesis.Total cellular RNA was isolated, and the remaining IL-3 mRNA determinedby Northern analysis. The half-life of IL-3 mRNA from anti-CD3 plusanti-CD28 treated cells was at least 8-fold longer than the IL-3 mRNAfrom anti-CD3 treated cells (Table 4). Thus, as would be expected fromthe previously described results of anti-CD28 on other lymphokines, itcan be concluded that the effect of anti-CD28 on IL-3 gene inductioncan, in large part, be explained by the ability of anti-CD28 tostabilize the IL-3 messenger RNA. TABLE 4 IL-3 GENE EXPRESSION(arbitrary densitometry CONDITION units) EXPERIMENT #1 CD28⁺ T cells, 6h, anti-CD3 1 CD28⁺ T cells, 6 h, anti-CD3 + anti-CD28 3-5 EXPERIMENT #2CD28⁺ T cells, 8 h, PMA 3 ng/ml + lonomycin >10 0.4 μg/ml CD28⁻ T cells,8 h, PMA 3 ng/ml + lonomycin <1 0.4 μg/ml EXPERIMENT #3 CD28⁺ T cells,anti CD3, then 1 actinomycin D 90 m CD28⁺ T cells, anti-CD3 + anti-CD28,8 then actinomycin D 90 m

SPECIFIC EXAMPLE IV Comparison of CD28 Stimulation to Stimulation ofOther T Cell Surface Molecules

[0100] CD28⁺ T cells were cultured at approximately 1×10⁵ cells/well inRPMI media containing 5% heat-inactivated fetal calf serum (FCS), PHA 10pg/ml, PMA 3 ng/ml, ionomycin at 100 ng/ml, anti-CD28 mAb 9.3 at 100 atng/ml, or mAb 9.4 specific for CD45 at 1 μg/ml or mAb 9.6 specific forCD2 at 1 μg/ml, or immobilized mAb G19-4 specific for CD3 at 200ng/well.

[0101] CD28⁺ T cells were cultured in quadruplicate samples inflat-bottomed 96-well microtiter plates in RPMI media containing 5%heat-inactivated fetal calf serum. Equal aliquots of cells were culturedfor 18 h and then pulsed for 6 h with 1 μCi/well of ³H-uridine, or for72 h and then pulsed for 6 h with 1 μCi/well of ³H-thymidine. The meansand standard deviations (in cpm) were determined by liquid scintillationcounting after cells were collected on glass fiber filters.

[0102] All cultures containing cells immobilized to plastic by anti-CD3monoclonal antibodies were visually inspected to ensure complete cellharvesting. The failure of cells in these cultures to proliferate inresponse to PHA is the result of rigorous depletion of accessory cells,in vivo activated T cells, B cells, and CD11⁺ (CD28⁻) T cells bynegative immunoabsorption as described in Specific Example II above. Ineach experiment, cells were stained with fluorescein-conjugated anti-CD2mAb OKT11 and fluorescein-conjugated anti-CD28 mAb 9.3 and were shown tobe over 99% and over 98% surface positive, respectively.

[0103] A representative experiment is illustrated in FIGS. 1 and 2. Asshown in FIGS. 1 and 2, anti-CD28 by itself had no significant effect onuridine or thymidine incorporation, nor did it serve to augmentproliferation induced either by immobilized anti-CD3 mAb G19-4 orchemically-induced T cell proliferation involving phorbol myristateacetate (PMA) and ionomycin (lono). However, as shown in FIG. 2,anti-CD28 did significantly increase the uridine incorporation of bothsets of cells. In contrast, other monoclonal antibodies includinganti-CD2 mAb OKT11 and anti-CD45 mAb 9.4 had no significant effect onuridine incorporation of anti-CD3 stimulated cells. This was not due tolack of effect of these antibodies on the cells, since anti-CD2monoclonal antibodies significantly augmented the proliferation ofanti-CD3 stimulated cells. In separate experiments, the binding ofisotype-matched mAbs to other T cell surface antigens (CD4, CD6, CD7 orCD8) failed to mimic the effects observed with anti-CD28.

[0104] These data serve to confirm that the stimulation of activated Tcells by CD28 has a unique phenotype which appears to directly enhancethe rate of incorporation of a radioactive marker into the steady stateRNA of T cells without directly enhancing T cell proliferation.

SPECIFIC EXAMPLE V

[0105] Increased Cellular Production of Human T_(H)CD28 Lymphokines byCD28 Stimulation Ex Vivo

[0106] Based on evidence from the in vitro systems it appeared that CD28did not have a significant effect on cellular production of lymphokinesunless they had undergone prior antigen activation or its equivalent.However, CD28 binding by the 9.3 mAb significantly enhanced the abilityof anti-TCR/CD3 activated T cells to sustain production of humanT_(H)1-type lymphokines. To test this effect in a physiologic setting,the activation of T lymphocytes in an ex vivo whole blood model wasstudied.

[0107] 50-100 ml of venous blood was obtained by standard asepticprocedures from normal volunteers after obtaining informed consent. Theblood was heparinized with 25 U/ml of preservative-free heparin(Spectrum, Gardenia, Calif.) to prevent clotting. Individual 10 mlaliquots were then placed on a rocking platform in a 15 ml polypropylenetube to maintain flow and aeration of the sample.

[0108] To assay for the effectiveness of CD28 stimulation on theinduction of lymphokine gene expression, the production of TNF-αmolecule was chosen as a model because of the extremely short half-life(approximately 15 minutes) of the protein in whole blood. 10 ml of wholeblood isolated as described above was incubated with soluble anti-CD3mAb G19-4 at a concentration of 1 μg/ml or anti-CD28 mAb 9.3 at aconcentration of 1 μg/ml or a combination of the two antibodies. Theplasma was assayed for TNF-α as described in Specific Example III at oneand four h. An example of one such experiment is shown in Table 5, whichillustrates the significant increase in sustained production of TNF-α bymaximal stimulation of CD3 and costimulation of CD28. TABLE 5 TNF-α(pg/ml) STIMULUS 0 h 1 h 4 h anti-CD3 4.5^(a) 65.0 2.1 anti-CD28 4.5^(a)1.6 3.3 anti-CD3 + anti-CD28 4.5^(a) 35.0 75.0

SPECIFIC EXAMPLE VI Resistance of CD28-Induced T Cell Proliferation toCyclosporine

[0109] The protocol used and results described herein are described indetail in June, C. H. et al., Mol. Cell. Biol., 7:4472-4481 (1987),herein incorporated by reference.

[0110] T cells, enriched by nylon wool filtration as described byJulius, et al., Euro. J. Immunol., 3:645-649 (1973), were cultured atapproximately 5×10⁴/well in the presence of stimulators in the followingcombinations: anti-CD28 mAb 9.3 (100 ng/ml) and PMA 1 (ng/ml); orimmobilized anti-CD3 mAb G19-4 (200 ng/well); or PMA (100 ng/ml). Theabove combinations also included fourfold titrations (from 25 ng/ml to1.6 μg/ml) of cyclosporine (CSP) (Sandoz, Hanover, N.J.) dissolved inethanol-Tween 80 as described by Wiesinger, et al., Immunobiol.,156:454-463 (1979).

[0111]³H-thymidine incorporation was measured on day 3 of culture andthe results representative of eight independent experiments are depictedin FIG. 3. The arithmetic mean (91,850±1300 (mean±SD)) CD28-induced Tcell proliferation exhibits nearly complete cyclosporine resistance whenaccompanied by the administration of PMA. Table 6 below illustrates theeffects of cyclosporine on CD3-induced proliferation of CD28⁺ T cellscultured at approximately 5×10⁴ cells/well in flat-bottomed 96-wellmicrotiter plates (CoStar, Cambridge, Mass.) under the followingconditions: immobilized Mab G19-4; or immobilized mAb G19-4 and mAb 9.30100 ng/ml; or immobilized mAb G19-4 and PMA 1 ng/ml; or Mab 9.3 100ng/ml and PMA 1 ng/ml. Cyclosporine was prepared as above and includedin the cultures at 0, 0.2, 0.4, 0.8, 1.2 μg/ml.

[0112]³H-thymidine incorporation was determined on day 3 of culture asabove. The percent inhibition of proliferation was calculated betweenCD28⁺ T cells cultured in medium only or in cyclosporine at 1.2 μg/ml.CD28⁺ T cells cultured in the absence of cyclosporine were givencyclosporine diluent. ³H-thymidine incorporation of cells cultured inmedium, or PMA, or monoclonal antibody 9.3 only were less than 150 cpm.As shown in Table 6, costimulation of CD3 and CD28 resulted in a markedincrease in the resistance of T cell proliferation to cyclosporine andthe stimulation of CD28 in the presence of PMA resulted in a completeabsence of cyclosporine suppression of T cell proliferation. As shown inTable 7, stimulation of CD28 together with immobilized anti-CD3 alsoresulted in resistance to suppression of T cell proliferation by theimmunosuppressant dexamethasone. TABLE 7 Effects of CD28 Stimulation onDexamethasone Resistance on T cell Proliferation ³HTdR + Dexamethasone(Nm) STIMULUS 0 25 % INHIBIT CD3 mAb G194 14,700 770 97 CD3 mAb + IL-221,700 1,900 93 CD28 mAb + PMA 181,600 197,700 <0 PMA 5,000 1,400 72

[0113] TABLE 6 Effects of CD28 Stimulation on Cyclosporine Resistance onT Cell Proliferation Mean [³H]thymidine incorporation (kcpm) ± 1 SD atCyclosporine Conc (ug/ml) STIMULUS 0 0.2 0.4 0.8 1.2 % INHIBIT CD3 mAbG19-4  77 ± 26 61 ± 6.8  52 ± 4.4 10 ± 3.4 8.2 ± 1.2  90 CD3 + CD28 123± 18 86 ± 2.3  63 ± 4.4 44 ± 6.4 43 ± 5.2 65 mAb 9.3 CD3 + PMA 145 ± 12132 ± 2.8  123 ± 6.4 55 ± 3.6 56 ± 6.4 62 CD28 mAb 111 ± 12 97 ± 5.6 107± 12  99 ± 14  112 ± 2.4  <0 9.3 + PMA

SPECIFIC EXAMPLE VII Human T_(H)CD28 Lymphokine Secretion in thePresence of Cyclosporine

[0114] As described in Specific Example III, CD28⁺ T cells were culturedin the presence of various stimulators. Culture supernatants wereharvested at 24 h and serial dilutions assayed for IL-2, TNF-α/LT,IFN-γ, and GM-CSF as previously described. Separate aliquots of cellswere recovered 48 h after stimulation and assayed for the percentage ofcells in late stages of the cell cycle (S+G₂+M).

[0115] When cyclosporine at 0.6 μg/ml was included in the test protocol,as shown in Table 8 (which also incorporates the data of SpecificExample III for comparison), CD28⁺ T cells were found to secrete theT_(H)CD28 lymphokines in the presence of cyclosporine in culturesstimulated with mAb 9.3 and PMA; or immobilized mAb G19-4 and mAb 9.3;or PMA and ionomycin and mAb 9.3. T_(H)CD28 lymphokine productioninduced by immobilized mAb G19-4; or by PMA with ionomycin was, however,completely suppressed in the presence of cyclosporine. TABLE 8 GM- S +IL-2 TNF-α/LT IFN-γ CSF G₂ + M STIMULUS (U/ml) (U/ml) (U/ml) (U/ml) (%)Medium <2 0 0   0 4.6 PMA <2 0 0 NT 5.5 anti-CD28 <2 5 0   0 6.5anti-CD28 + PMA 435 300 24  150 48.9 anti-CD28 + PMA + 192 200 12 NT49.3 CSP anti-CD3^(i) 36 50 24  120 39.7 anti-CD3^(i) + CSP <2 0 0 NT14.5 anti-CD3^(i) + anti- 1200 40 74 1050 44.7 CD28 anti-CD3^(i) + anti-154 200 9 NT 48.6 CD28 + CSP lonomycin <2 0 0 NT 6.6 lonomycin + PMA 2005 37 NT 43.6 lonomycin + PMA + <2 0 0 NT 8.1 CSP lonomycin + PMA + 1640320 128 NT 43.5 anti-CD28 lonomycin + PMA + 232 120 15 NT 47.6anti-CD28 + CSP

SPECIFIC EXAMPLE VIII Human T_(H)CD28 Lymphokine mRNA Expression in thePresence of Cyclosporine

[0116] In order to further examine whether CD28 stimulation led tocyclosporine-resistant T_(H)CD28 lymphokine gene expression as well assecretion, the ability of cyclosporine to suppress induction of IL-2,TNF-α, LT, INF-γ, and GM-CSF following stimulation by variousstimulators was tested. Specifically, CD28⁺ T cells were cultured at2×10⁶/ml in complete RPM1 medium (GIBCO, Grand Island, N.Y.) with 5% FCS(MED). Individual aliquots of CD28⁺ T cells were incubated for 6 h inthe presence or absence of 1.0 μg/ml cyclosporine with PMA 3 ng/ml andanti-CD28 mAb 9.3 (1 mg/ml); or with immobilized anti-CD3 mAb G19-4 (1μg/well); or with immobilized mAb G19-4 (1 μg/well) and mAb 9.3 (1ng/ml). CD28⁺ T cells were harvested, total cellular RNA isolated andequalized for ribosomal RNA as previously described by Thompson, et al.,Nature, 314:363-366 (1985).

[0117] Northern blots were prepared and hybridized sequentially with³²P-labeled, nick-translated gene specific probes as described by June,C. H. et al., Mol. Cell. Biol., 7:4472-4481 (1987). The IL-2 probe was a1.0 kb Pst I cDNA fragment as described by June, C. H. et al., Mol Cell.Biol., 7:4472-4481 (1987); the IFN-γ probe was a 1.0 kb Pst I cDNAfragment as described by Young, et al., J. Immunol., 136:4700-4703(1986). The GM-CSF probe was a 700 base pair EcoR I-Hind III cDNAfragment as described by Wong, et al., Science, 228:810-815 (1985); the4F2 probe was a 1.85 kb EcoR I cDNA fragment as described by Lindsten,et al., Mol. Cell. Biol., 8:3820-3826 (1988); the IL4 probe was a 0.9 kbXho I cDNA fragment as described by Yokota, et al., PNAS (USA),83:5894-5898 (1986); and the human leukocyte antigen (HLA) probe was a1.4 kb Pst I fragment from the HLA-B7 gene as described by Lindsten, etal., Mol. Cell. Biol., 8:3820-3826 (1988). TNF-α and LT specific probeswere synthesized as gene-specific 30 nucleotide oligomers as describedby Steffen, et al., J. Immunol., 140:2621-2624 (1988) and Wang, et al.,Science, 228:149-154 (1985). Following hybridization, blots were washedand exposed to autoradiography at −70° C. Quantitation of band densitieswas performed by densitometry as described in Lindsten, et al., Mol.Cell. Biol., 8:3820-3826 (1988).

[0118] As illustrated by the Northern blot of FIG. 4, stimulation by mAb9.3 with PMA and by mAb 9.3 with mAb G19-4 led to human T_(H)CD28lymphokine gene expression that exhibited resistance to cyclosporine. Incontrast, stimulation by TCR/CD3 mAb G19-4 alone was completelysuppressed in the presence of cyclosporine.

SPECIFIC EXAMPLE IX In Vivo Activation of T Cells by CD28 Stimulation

[0119] A. Monoclonal Antibody 9.3 F(ab′)₂.

[0120] F(ab′)₂ fragments of mAb 9.3 were prepared as described byLedbetter, J. A. et al., J. Immunol., 135:2331-2336 (1985). Purified andendotoxin-free F(ab′)₂ fragments were injected intravenously at 1 mg/kgof body weight over a 30 minute period into a healthy macaque (M.nemestrina) monkey. On days 2 and 7 after injection, 5 ml of blood wasdrawn and tested.

[0121] Peripheral blood lymphocytes from the monkey's blood wereisolated by density gradient centrifugation as described in SpecificExample II. Proliferation of peripheral blood mononuclear cells inresponse to PMA (1 ng/ml) was tested in the treated monkey and a controlanimal (no F(ab′)₂ fragment treatment) in triplicate as described inSpecific Example IV. Proliferation was measured by the uptake of³H-thymidine during the last 6 h of a three-day experiment and theresults shown in FIG. 5. Means of triplicate culture are shown, andstandard errors of the mean were less than 20% at each point. As shownin FIG. 5, in vivo stimulation of CD28 by the F(ab′)₂ mAb 9.3 fragmentincreased T cell proliferation for at least 7 days.

[0122] B. Monoclonal Antibody 9.3

[0123] Two doses of mAb 9.3, 10 mg and 0.1 mg, were administeredintravenously to primates Macaca mulatta. The antibody wa infusedintravenously immediately after baseline (time zero) blod valves wereobtained. Three animals were evaluated as described below at each dose.

[0124] Cell population changes. At the higher dose, immediate effectswere monitored over the first 120 m. In FIGS. 6A and 6B, arepresentative result is depicted showing the change in the lymphocytecounts over time. The ALC and distribution of CD28⁺ cells are depictedin FIG. 6A, while FIG. 6B illustrates the absolute numbers of CD4⁺ andCD8⁺ cells. The absolute lymphocyte count (ALC) decreased over the first60 m and then increased above baseline at 24 h (see FIG. 6A). In thiscase, the number of circulating CD28 positive lymphocytes remainedessentially the same, as determined by adding goat anti-mousephycoerythrin (GAM-PE) only or mAb 9.3 plus GAM-PE. In this same animal,the CD4 and CD8 positive populations were followed and the increase at24 h was the result of an increase in CD8⁺CD28⁻ cells (see FIG. 6B).Animals re-evaluated after 8 days had between 35 to 60% of the CD28⁺cells coated with antibody. There was no significant change in thecirculating lymphocyte counts in primates treated with 0.1 mg mAb 9.3.

[0125] Cytokine released after in vitro stimulation. PBLs isolated atspecific time points from primates previously immunized with tetanustoxoid and treated with 10 mg mAb 9.3 were cultured in vitro todetermine the effect of antigenic stimulation on cytokine production.FIG. 7A represents the in vitro production of TNF while FIG. 7Brepresents the in vitro production of IL-6. PBLs stimulated withConcanavalin-a (Con-a) are depicted by Δ. PBLs stimulated with tetanustoxoid (TT) are depicted by . Unstimulated PBLs are depicted by ∘. Asshown in FIGS. 7A and 7B, cultures of baseline cells did not respond toeither Con-a or TT stimulation. However, as shown in FIG. 7A, PBLsisolated from animals 6 h after infusion of antibody showed an increasein TNF production. As depicted in FIG. 7B, after 24 h, unstimulatedcultures produced TNF but not IL-6. TT stimulation of PBLs produced asimilar quantity of both TNF and IL-6 as mitogen stimulated cultures.This response was consistent through 72 h.

[0126] Monoclonal antibody 9.3 was administered to the primates eitheras a single day bolus of 10 mg (n=3) or as multiple daily injections of10 mg/d for 5 consecutive days (n=3) and the animals were followedsimultaneously. The changes in the peripheral blood cell populationswere not dramatic. ALC as previously observed decreased with the firstinjection but recovered to above baseline if no further injections wereadministered. However, for animals treated with multiple injections, ALCremained ˜25% below normal during the period of mAb administration. ALCin multiple-treated animals did not recover to above normal levels overthe 21-day study period. Absolute neutrophil count decreased by ˜30% inthe 5 day treated group.

[0127] Cytokine levels in serum. Serum was analyzed for IL-6, TNF, andIL-1 β. The detection of TNF from the serum preparations was notsuccessful and therefore no results are available at this time. FIGS. 8Aand 8B demonstrate the serum concentration of IL-6 after infusion of mAb9.3. As shown in FIGS. 8A and 8B, increased IL-6 levels 24 h after mAbinfusion were detected. In animals injected one time, the IL-6 levelsincreased to a peak on day 4, but a decrease was observed whenremeasured on day 8 (see FIG. 8A). In comparison, 5 day treated animals(multiple doses) demonstrated continual increase(s) in IL-6 through day8 (see FIG. 8B).

[0128]FIGS. 9A and 9B demonstrate the serum concentration of IL-1 afterinfusion of mAb 9.3. As shown in FIGS. 9A and 9B, measurements of IL-1 βin the serum, did not detect any IL-1 until after day 8 in singleinjected animals (see FIG. 9A) or multiple injected animals (see FIG.9A). The multiple injected animals, however, had increasing levels ofIL-lβ at day 21 post-infusion, while single injected animals haddecreasing levels at this time.

[0129] Cytokine release after in vitro stimulation. IL-6 production wasnot detected in the PBLs of animals on day 3 after in vitro stimulationwith TT. (FIG. 10). This finding contrasted with the previous in vitroresults. However, an increased production of IL-6 was detected on day 7and the more significant increase was observed from PBLs isolated fromthe 5 day treated primates. This increase in production was furtherobserved in culture of PBLs isolated on day 14.

[0130] IL-6 Production and Proliferation of PBLs. FIGS. 10A and 10Billustrate IL-6 production of in vitro stimulated PBLs isolated frommonkeys. Days 1, 3 and 14 are depicted in FIGS. 10A and 10B with Δrepresenting the control and ∘ representing the stimulated PBL response.FIG. 10A illustrates the response of a single injected animal and FIG.10B illustrates the response of a multiple injected animal. (Note thatthe quantity of cells harvested from PBL limited the number of assaysperformed, resulting in no day zero points and no day zero data.) PBLswere isolated from the different treated groups and evaluated for theirproliferative response to Con-A, TT or no stimulus. Historically the TTresponse was ˜5,000 cpm and the baseline Con-A response was ˜35,000 cpm.The PBL proliferative response to Con-A was reduced by about 80% andgradually recovered over time (not shown). No proliferative response wasobserved when the PBL were stimulated with TT. This contrasts with thelymphokine production observed in in vitro cultures.

SPECIFIC EXAMPLE X Immunoregulation with CTLA-4lg

[0131] A. In Vitro.

[0132] The effects of CTLA-4lg on the primary immune response toalloantigen was initially examined in a one-way mixed lymphocyte culture(MLC) between Lewis rats (RT1^(l), responder) and Brown-Norway rats(RT1^(n), stimulator). Lymphocytes were isolated from paratracheal andcervical lymph nodes. Cultures were performed in quadruplicate in96-well round bottomed plates as described in Turka, L. A. et al.,Transplant., 47:388-390 (1989). Cultures were harvested after 4 days and1 mCi/well of ³H-thymidine was added for the last 6 h of culture. Inthis assay, Brown-Norway stimulator cells were irradiated at 30 Gy toprevent their proliferation, and then added to cultures of Lewisresponder lymphocytes. A proliferative response will normally occur inapproximately 1-5% of cells as a result of activation through theircell-surface TCR in response to allogeneic MHC as discussed in Marrack,P. et al., Immunol. Today, 9:308-315 (1988). Graded concentrations ofCTLA-4lg or an isotype-matched control monoclonal antibody L6 describedin Fell, H. P. et al., J. Bio. Chem. (in press), was added to thecultures.

[0133]FIG. 11 represents the effect of CTLA-4lg on a one-way mixedlymphocyte culture. Spontaneous proliferation is the incorporation ofthymidine by Lewis cells in the absence of Brown-Norway stimulators, andis depicted by closed triangles in FIG. 11. As shown in FIG. 11,CTLA-4lg was able to block proliferation in a dose dependent fashionwith virtually complete inhibition observed at a concentration of 1mg/ml. (Results are expressed as counts per minute of ³H-thymidineincorporation±standard deviation). Consistent with these results,alloreactive T cell responses can also be inhibited by non-stimulatoryFab fragments of an anti-CD28 monoclonal antibody as shown in Azuma, M.et al., J. Exp. Med., 175:353-360 (1992). Together, these data suggestthat in order to mount a proliferative response in vitro, alloreactive Tcells must be stimulated not only through MHC engagement of the TCR butalso require costimulation by B7 engagement of the CD28 receptor.

[0134] B. In Vivo: Cardiac Allografts.

[0135] CTLA-4lg was next used in a rat model of organ transplantation toascertain its ability to block alloantigen responses in vivo. Recipientanimals received a heterotopic cardiac allograft which was anastomosedto vessels in the neck as described in Bolling, S. F. et al.,Transplant., 53:283-286 (1992). Grafts were monitored for mechanicalfunction by palpation and for electrophysiologic function byelectrocardiogram. Graft rejection was said to occur on the last day ofpalpable contractile function. As an initial test, animals were treatedwith daily injections of CTLA-4lg or an isotype-matched negative controlmonoclonal antibody L6 for 7 days. CTLA-4lg was administered at doses of0.015 mg/day (5 animals), 0.05 mg/day (5 animals), and 0.5 mg/day (8animals). L6 was given at 0.5 mg/day. Untreated Lewis rats rejected theheterotopic Brown-Norway allografts in 6.8±0.3 days (n=10). Theallografts in CTLA-4lg-treated animals remained functional followingcompletion of drug administration, whereas untreated animals, or animalstreated with the L6 control antibody, uniformly rejected their grafts byday 8 (p<0.0001) as shown in Table 9. (p values were calculated byChi-square analysis). TABLE 9 GRAFT SURVIVAL DAY 8 SIGNIFICANCEUntreated  0/10 p < 0.0001 CTLA-4lg 18/18 p < 0.0001 Control Protein 0/5

[0136] CTLA-4lg-treated rats manifested no observable acute or chronicside effects from administration of the protein. No gross anatomicabnormalities were observed in CTLA-4lg-treated animals at autopsy.

[0137] An untreated animal and a CTLA-4lg-treated animal were sacrificedfor histological examination. Cardiac allografts were removed from anuntreated animal (shown in FIG. 12A) and a CTLA-4lg-treated animal (0.5mg/day) (shown in FIG. 12B) four days after transplantation. Allograftswere fixed in formalin, and tissue sections were stained withhematoxylin-eosin. (Original photography at 200× magnification.) Thedonor heart removed from the untreated animal showed histologicalfindings of severe acute cellular rejection, including a prominentinterstitial mononuclear cell infiltrate with edema formation, myocytedestruction, and infiltration of arteriolar walls. In contrast, thetransplanted heart from the CTLA-4lg-treated animal revealed only a mildlymphoid infiltrate. Frank myocyte necrosis and evidence of arteriolarinvolvement were absent. The native heart from each animal showed nohistological abnormalities.

[0138] To determine whether CTLA-4lg therapy established a state ofgraft tolerance that persisted following drug treatment, animals treatedfor 7 days with daily injections of CTLA-4lg were observed withoutadditional therapy until cessation of graft function. Animals receivedeither no treatment, CTLA-4lg (0.5 mg/day×7 days), or an isotype-matchedcontrol monoclonal antibody, L6 (0.5 mg/day×7 days). In all casestreatment was initiated at the time of transplantation. FIG. 13 is agraph showing allograft survival in the treated and control rats. Graftsurvival was 18-40 days in animals treated with 0.05 mg/day of CTLA-4lg.Graft survival was assessed daily. This failure to induce permanentengraftment did not appear to be due to inadequate dosing of CTLA-4lg,as animals treated with a ten-fold higher dose, 0.5 mg/day, showed asimilar graft survival curve as depicted in FIG. 13, with one animalmaintaining long-term graft function (>50 days). In FIG. 13, graftsurvival is displayed as the last day of graft function. Animals treatedwith a dose of 0.015 mg/day×7 days (n=5) had a mean survival of 12.6±2.1days (n=5). Furthermore, serum CTLA-4lg trough levels in this group asmeasured in a quantitative ELISA assay were in excess of 10 μg/ml, aconcentration which is maximally suppressive in vitro (see FIG. 11).Histological examination of the allografts from CTLA-4lg-treated animalswhose grafts ceased functioning after 18-43 days displayed typical signsof acute cellular rejection, of the same degree of severity as seen incontrol animals that had rejected their hearts after 7 days. The animalwith continued graft function was sacrificed on day 57, and theallograft from this animal failed to reveal any histologicalabnormalities.

[0139] At the time of sacrifice, lymphocytes from the day 57 “tolerant”animal, and from a CTLA-4lg-treated animal that rejected the heart atday 33, were tested for their functional responses. These responses werecompared with those of lymphocytes from a control (non-transplanted)Lewis rat, and results were normalized as a percentage of the controlresponse. In comparison to control animals, lymphocytes from both the“tolerant” and rejecting animal had equivalent proliferative responsesto Con-a, (tolerant, 62.5%; rejecting, 51.1%; p=0.63, two-tailed T test)and to cells from a third party ACI rat (RT1^(avl)) (tolerant, 160%;rejecting, 213%; p=0.58). However a significant disparity was seen inthe response to Brown-Norway cells (tolerant, 34.2%; rejecting, 238%;p<0.005), suggesting that T cells from animals with functioning graftswere specifically hyporesponsive to donor MHC antigens. The thymus andspleen from the day 57 “tolerant” animal were similar in size and cellnumber to the non-transplanted control rat, and flow cytometric analysesof thymus, lymph nodes and spleen revealed similar percentages of bothCD4⁺ and CD8⁺ T cells in each animal. Splenocytes adoptively transferredfrom the day 57 “tolerant” animal into a native Lewis recipient failedto affect the rate at which that animal rejected a Brown-Norway cardiacallograft. Thus, tolerance did not appear to be maintained by suppressorcells in this animal.

[0140] The fact that 4 of 5 allograft recipients treated with high doseCTLA-4lg (0.5 mg/day×7 days) rejected their grafts within the studyperiod indicated that blockade of the costimulatory molecule B7 did notconsistently induce permanent graft tolerance. One possible explanationwas that CTLA-4lg induced a temporary state of non-responsiveness, andthat upon recovery, recipient T cells could effect graft rejection.Alternatively, CTLA-4lg treatment may have resulted in a state ofpermanent non-responsiveness in circulating T cells by allowing targetantigen recognition without B7-dependent costimulation. Newly matured Tcells emerging from the thymus after cessation of CTLA-4lg treatmentcould not be tolerized by this mechanism, and could mediate graftrejection as a result of B7-costimulated T cell alloreactivity. Todifferentiate between these two possibilities, rats were thymectomized 3days prior to cardiac transplantation, and treated with daily injectionof CTLA-4lg (0.5 mg/day×7 days) following transplantation. These animalsrejected their grafts between days 28 and 33, indicating that allograftrecipients were not dependent upon the influx of new T cells to initiatean alloimmune response. Thus, it appears that T cells present during thetime of CTLA-4lg treatment can eventually induce graft rejection. Thismay represent T cell recovery from a temporary state ofnon-responsiveness, or may reflect the kinetics of T cell traffickingduring the CTLA-4lg treatment period.

[0141] C. Synergistic Effects with Cyclosporine.

[0142] Based on the ability to show that a soluble CD28 receptorhomologue, CTLA-4lg, is capable of suppressing cell-mediated responsesin vitro and in vivo, experimentation was performed to determine whetheror not this immunosuppressant has additive or synergistic effect withcyclosporine. A mixed lymphocyte reaction (MLR) with Brown-Norway ratlymph node cells as stimulators and Lewis strain rat lymph node cells asresponders was measured by measuring tritiated thymidine incorporation72 h after cocultivation. The ability of CTLA-4lg at a concentration of0.1 μg/ml and cyclosporine at 30 ng/ml alone or in combination wasmeasured. FIG. 14 shows ³H-thymidine incorporation under variousconditions. As can be seen in FIG. 14, although either immunosuppressantled to only a partial reduction in the mixed lymphocyte proliferativeresponse (MLR+CTLA-4lg and MLR+CSP), the combination of the two(MLR+CSP+CTLA-4lg) completely blocked the mixed lymphocyte reactionbetween these MHC-incompatible strains. This effect is greater than whatwould be expected from two immunosuppressive reagents which haveadditive effects, suggesting that CTLA-4lg and cyclosporine block T cellactivation by independent mechanisms and have a synergistic effect on Tcell activation in response to alloantigens.

SPECIFIC EXAMPLE XI Control of Lymphokine Production by SecondMessengers

[0143] A variety of second messengers in the regulation of lymphokineproduction were examined. In particular, a role for the two primary cellsecondary messenger systems, the activation of protein kinase C andelevation in intracellular calcium, were characterized as being centralregulators of the transcription of lymphokine genes. In addition,specific tyrosine phosphorylation events were identified that maycorrelate with the generation of alterations in translation and/or MRNAstability. Further investigations into serine and threonine kinasesindicate that they may also have a role in the signal transductionevents involved in lymphokine production. In contrast, experiments intothe regulation by cGMP showed that this agent has relativelynon-specific effects on lymphokine production.

[0144] Tyrosine phosphorylation events related to CD28 were furtherstudied as described below.

Protocol

[0145] Monoclonal antibodies. Anti-CD2 mAb G19-4 (IgG1), anti-CD28 mAb9.3 (IgG2a), anti-CD5 mAb 10.2 (IgG2a), and anti-CD45 mAb 9.4 (IgG2a)were produced, purified and in some cases, biotinylated as described inLedbetter, J. A. et al., J. Immunol., 135:2331 (1985) and Ledbetter, J.A. et al., J. Immunol., 137:3299 (1986). Anti-B7 mAb 133-(IgM) anddilutions of ascites as described in Freedman, A. S. et al., J.Immunol., 139:3260 (1987), were used. Anti-CD3 mAb OKT3 (IgG2a) wasabsorbed to goat anti-mouse IgG covalently linked to microspheres (KPL,Gaithersburg, Md.), by incubation of a 1/10⁵ dilution of pooled asciteswith 10⁷ beads/ml in HBSS at room temperature, followed by extensivewashing.

[0146] Cells. The CD28⁺ subset of T cells was isolated from peripheralblood T lymphocytes by negative selection using immunoabsorption withgoat anti-mouse lg-coated magnetic particles as previously described inJune, C. H. et al., Mol. Cell. Biol., 7:4472 (1987). This resulted in apopulation of resting T cells that was >99% CD3⁺ and that did notcontain CD2⁺/CD3⁻ cells such as NK cells. The Jurkat T leukemia cellline E6-1 was a gift from Dr. A. Weiss and maintained in complete media,i.e. RPMI 1640 containing 2 mM L-glutamine, 50 μg/ml gentamycin, and 10%FCS (HyClone Laboratories, Logan, Utha). In some instances, T cells orJurkat cells were cultured in complete media, or in complete media with5 ng/ml PMA (Sigma Chemical Co., St. Louis, Mo.) or OKT3 beads (±15beads/cell) before experiments. The Jurkat J32 cell line (CD2⁺, CD3⁻,CD28⁺) has been described in Makni, H. et al., J. Immunol., 146:2522(1991). J32 variants (CD2⁺, CD3⁻, CD28⁺) were derived by γirradiation-induced mutagenesis and immunoselection (see Makni, supra(1991)); one such cloned mutant, J32-72.4 is stable in culture. Thesurface receptor expression of these cells was quantitated by indirectimmunofluorescence and analyzed by flow cytometry. The mean logfluorescence intensity for each sample was determined and was convertedinto linear relative fluorescence units (ΔFL) by the formulaΔFL=10^([(E-C)/D]); where E is the mean log fluorescence intensity ofthe experimental antibody sample, C is the mean log fluorescenceintensity of the control antibody sample, D is 50 channels/decade. Forthe TCR/CD3 and CD28 receptors, ΔFL of the J32 cells was 27.0 and 57.0,and for the J32-74.2 cells 1.1 and 40.7. Northern blot analysis ofJ32-72.4 revealed no detectable TCR-β mRNA, while the expression of theTCR-α, CD3-γ, δ, and ε and TCR ζ mRNA was similar to that of theparental J32 cells (unpublished data).

[0147] B7 transfection of CHO cells. CHO cells were transfected with B7cDNA as previously described in Gimmi, C. D. et al., PNAS (USA), 88:6575(1991). These cells have previously been shown to stimulate lymphocyteproliferation and lymphokine secretion in a manner that mimics CD28mAb-induced T cell activation. See Linsley, P. S. et al., J. Exp. Med.,173:721 (1991) and Gimmi, supra (1 991). Transfected CHO cells showingno B7 expression were recloned and are referred to as CHO-B7⁻. CHO cellswere detached from tissue culture plates by incubation in PBS with 0.5mM EDTA for 30 m and fixed in 0.4% paraformaldehyde as described inGimmi, supra (1991). Fixed CHO-B7⁻ cells were used as control cells.

[0148] Immunoblot analysis of protein tyrosine phosphorylation. Detailsof the immunoblot assay with anti-phosphotyrosine antibodies has beendescribed in Hsi, E. D. et al., J. Biol. Chem., 264:10836 (1989) andJune, C. H. et al., J. Immunol., 144:1591 (1990). Cells were suspendedat 5-10×10⁷ cells/ml in reaction media, i.e., HBSS containing 0.8% FCSand 20 Mm Hepes at 37° C. at time −3 m and stimulated at time 0 m. mAbswere used at 10 μg/ml final concentration. For crosslinking,biotinylated mAbs were incubated with cells for 5-8 m at roomtemperature, the cells prewarmed at time 3 min and stimulated withavidin (Sigma Chemical Co.) at a final concentration of 40 μg/ml at time0. Stimulation was terminated by the addition of ice-cold 10× lysisbuffer, yielding a final concentration of 0.5% Triton X-100. See June,J. Immunol., supra (1990). After lysis at 4° C., nuclei were pelletedand postnuclear supernatants were subject to SDS-PAGE on a 7.5% gel,transferred to polyvinylidene difluoride microporous membrane(Millipore, Bedford, Mass.) and the membranes probed withaffinity-purified anti-phosphotyrosine antibodies, labeled with ¹²⁵Istaphylococcal protein A (ICN, Irvine, Calif.) and exposed to x-rayfilm.

Results

[0149] Herbimycin A prevents CD28-stimulated IL2 production. Previousstudies have shown that three distinct biochemical signals, provided byphorbol esters, calcium ionophore, and ligation of the CD28 receptorwith mAb, are required to cause optimal IL-2 secretion (see June, C. H.et al., J. Immunol., 143:153 (1989)). Cells cultured in the presence ofPMA, ionomycin, or CD28 mAb alone produced no detectable IL-2 and, aspreviously reported in June, J. Immunol., (1989) supra, and Fraser, J.D. et al., Science (Wash., D.C.) 251:313 (1991), stimulation of the CD28receptor strongly up-regulated IL-2 production of T cells stimulatedwith immobilized anti-CD2 mAb, PMA, or PMA plus ionomycin. To addressthe potential role of tyrosine kinases in CD28-triggered signaling, theeffect of herbimycin A, an inhibitor of the src family protein tyrosinekinases (see Uehara, Y. et al., Biochem. Biophys. Res. Commun., 163:803(1989)), on the CD28-triggered enhancement of IL-2 production wasinvestigated. T cells were cultured overnight in the absence (depictedas open bars in FIG. 15) or presence (depicted as filled bars in FIG.15) of herbimycin A (1 μM). The cells were then cultured for a further24 h period in the presence of medium-immobilized anti-CD3 mAb (G19-4),PMA (3 ng/ml) (P), or PMA plus ionomycin (150 ng/ml) (P+l) in thepresence or absence of soluble anti-CD28 mAb 9.3 (1 ug/ml). Cell-freesupernatant was collected, dialized to remove herbimycin A and serialdilutions were analyzed for IL-2 content by bioassay as described inJune, J. Immunol., supra (1989). FIG. 15 shows the effect of herbimycinA on CD28-stimulated IL-2 production. The CD28 mAb mediated enhancementof IL-2 production in response to stimulation with immobilized anti-CD3,or PMA was nearly completely inhibited in the presence of herbimycin A.In contrast, cells cultured in PMA, ionomycin or 9.3 mAb only produced<10 U/mI of IL-2.

[0150] Disruption of the proximal signaling pathway triggered throughCD3 could potentially explain the effect of herbimycin on cellsstimulated with anti-CD3 and anti-CD28. Consistent with this,CD3-triggered IL-2 production was previously shown to be exquisitelysensitive to herbimycin A. See June, C. H. et al., PNAS (USA), 87:7722(1990). However, IL-2 production induced with the combination of PMAplus ionomycin or PMA plus CD28 stimulation permits, in principle, theability to isolate the CD28 signal for testing the effect of herbimycinA. PMA plus anti-CD28-stimulated IL-2 production was sensitive to theeffects of herbimycin A while, as previously noted, PMA plusionomycin-stimulated IL-2 secretion was resistant to the effects ofherbimycin A. The combination of PMA plus ionomycin plusanti-CD28-stimulation resulted in more IL-2 secretion than optimalamounts of PMA plus ionomycin, consistent with the previous reports ofJune, J. Immunol., supra (1989); and Fraser, J. D. et al., Science(Wash. D.C.) 251:313 (1991). However, in the presence of herbimycin A,PMA plus ionomycin plus CD28-stimulated cells produced approximatelyequivalent amounts of IL-2 as cells stimulated in the absence ofherbimycin with PMA plus ionomycin. Together, the above results suggestthat the function of both the TCR and CD28 receptors are sensitive toherbimycin, and further suggest the independent effects of these threereagents on IL-2 gene expression. See June, J. Immunol., supra (1989);and Fraser, supra (1991).

[0151] CD28 receptor crosslinking with mAb induces protein tyrosinphosphorylation in PMA-treated Jurkat cells. Given the above functionalresults, the potential involvement of protein tyrosine phosphorylationin CD28-mediated signal transduction was investigated by immunoblotanalysis of postnuclear supernatants of whole cell lysates of the T cellleukemia line Jurkat E6-1. Jurkat E-6 cells were cultured for 2 days inthe presence or absence of PMA (5 ng/ml). After washing, 10⁷ cells in120 μl were stimulated with reaction media (control), anti-CD3 Mab(G19-4), anti-CD28 mAb (9.3), or crosslinked anti-CD28 mAb (9.3) (finalconcentration, 10 μg/ml). For crosslinking, biotinylated mAb was addedat time 10 m, followed by avidin (40 μg/ml) at time zero. After 2 m, thereaction was terminated with ice-cold lysis buffer and postnuclearsupernatants were resolved by SDS-PAGE electrophoresis, transferred toimmobilon, and immunoblotted with antiphosphotyrosine, followed by¹²⁵I-protein A and autoradiography.

[0152] In a previous report by Ledbetter, J. A. et al., Blood, 75:1531(1990), increased tyrosine phosphorylation could not be detected inresting T cells after crosslinking the CD28 receptor. Consistent withthat report, no changes in tyrosine phosphorylation were detected inunstimulated Jurkat cells after the binding of bivalent or crosslinkedCD28 mAb. Previous studies have shown that CD28 stimulation alone doesnot result in lymphokine production in Jurkat cells or induceproliferation of primary T cells. See Weiss, A. et al., J. Immunol.,137:819 (1986); Martin, P. J. et al., J. Immunol., 136:3282 (1 986); andHara, T. et al., J. Exp. Med., 161:1513 (1985). Engagement of CD28 byCD28 mAbs or by B7, the natural CD28 ligand, delivers a costimulatorysignal provided T cells are stimulated with PMA or with TCR/CD3 mAbs.See June, C. H. et al., Immunol. Today, 11:211 (1990); Koulova, L. etal., J. Exp. Med., 173:759 (1991); Linsley, P. S. et al., J. Exp. Med.,173:721 (1991); and Gimmi, C. D. et al., PNAS (USA) 88:6575 (1991). Itthus appeared that CD28-induced protein tyrosine phosphorylation mightonly occur in the context of a costimulatory signal.

[0153] To test this hypothesis, Jurkat cells were cultured in PMA andthen stimulated with anti-CD28 mAb as previously described. In thePMA-stimulated cells, crosslinking of CD28 for 2 m inducedphosphotyrosine on substrates migrating with approximate molecularmasses of 47, 62, 75, 82, 100, 110, and 145 kD. Bivalent CD28 mAbinduced tyrosine phosphorylation, but to a lesser magnitude. Inagreement with June, C. H. et al., J. Immunol., 144:1591 (1990), CD3triggering of Jurkat cells induced tyrosine phosphorylation ofphosphoprotein (pp) 56, pp65, pp75, pp 100, pp110, and pp145 in restingJurkat cells and in PMA-treated Jurkat cells. Of particular interestwere pp75 and pp100, which were consistently phosphorylated by CD28stimulation under all conditions tested.

[0154] CD28 receptor crosslinking with Mab induces protein tyrosinephosphorylation in normal T cells. Similar experiments with highlypurified peripheral blood T cells from normal human donors wereperformed in order to determine if CD28 could increase tyrosinephosphorylation in nontransformed cells. Peripheral blood CD28⁺ T cellswere cultured in PMA (5 ng/ml) for 6 h. After washing, 10⁷ cells werestimulated for 2 m with media (control), anti-CD3 mAb (G19.4), anti-CD28mAb (9.3), crosslinked anti-CD28 mAb (9.3), or crosslinked anti-CD5 mAb(1 0.2). Cells were lysed and protein tyrosine phosphorylation wasdetermined as previously described. Crosslinking of CD28 on PMA-treatedcells induced the appearance of tyrosine phosphorylated substrates thatmigrated at 45, 75, and 100 kD. Again, pp75 and pp100 were mostprominent and consistently reproduced.

[0155] The effects of CD28 stimulation observed after 24-48 h of PMAstimulation were more pronounced than those seen after 6 h. Ligation ofCD28 by mAb on resting T cells caused the appearance of weakly detectedtyrosine phosphorylation. The induction of increased responsiveness toanti-CD28 mAb stimulation by PMA is slow in that 4-6 h of PMA treatmentare required to consistently observe CD28-induced tyrosinephosphorylation. Experiments with cycloheximide indicate that newprotein synthesis is required for cells to become responsive to CD28.The specificity of the CD28-induced tyrosine phosphorylation wasinvestigated by crosslinking CD5 with an isotype-matched mAb. Increasedtyrosine phosphorylation on the 75 kD substrate was occasionally inducedby CD5 crosslinking. In contrast, CD5 never induced tyrosinephosphorylation on pp100. Similarly, crosslinking of the MHC class Ireceptor also did not induce tyrosine phosphorylation of this substrate.

[0156] CD28 receptor crosslinking induces protein tyrosinephosphorylation in CD3-treated normal T cells. The above experimentssuggested that the CD28 receptor is relatively inactive in quiescentcells, and becomes responsive consequent to protein kinase C activation.To determine whether TCR stimulation could also prime cells for the CD28signal, T cells were cultured overnight in medium or in the presence ofanti-CD3-coated beads. The cells were recovered, and 8×10⁶ cells werestimulated with crosslinked anti-CD28 mAb for 0-5 m, the cells lysed,and protein tyrosine phosphorylation determined as previously described.Crosslinked CD28 mAb induced low level tyrosine phosphorylation onmultiple substrates in resting T cells that peaked 2-5 m after CD28stimulation. In contrast, CD28 mAb induced marked tyrosinephosphorylation in CD3-primed cells that was maximal within 1 m. Thus,costimulation of T cells with anti-CD3 augmented CD28-induced tyrosinephosphorylation as manifested by an increased magnitude of response andan accelerated kinetics of response. This induction of responsiveness toCD28 did not require DNA synthesis, as separate studies have shown thatthe T cell blasts used for these studies were in the late G₁ phase ofthe cell cycle.

[0157] CD28 receptor-B7/BB1 receptor interaction induces specifictyrosin phosphorylation in T cells. The above results indicate that CD28mAb can increase tyrosine phosphorylation in a variety of substrates onpreactivated T cells. Previous studies have indicated that CD28 appearsto deliver two biochemically distinct signals, depending on the degreeof crosslinking. See Ledbetter, J. A. et al., Blood, 75:1531 (1990). Theunique functional properties of CD28 mAb observed after stimulation of Tcells do not require highly crosslinked CD28 mAb and are obtained usingintact or F(ab′)₂ CD28 mAb. As discussed in Hara, T. et al., J. Exp.Med., 161:1513 (1985), studies have shown that CHO cells expressing theCD28 ligand mimic the functional effects of CD28 mAb. See Linsley, supra(1991), and Gimmi, supra (1991). These cells presumably represent a morephysiologic means to study CD28 receptor-mediated signal transduction.CHO-B7⁺ cells were incubated with PMA-treated T cells at a CHO/T cellratio of 1:10 for 5-30 m. B7-transfected CHO cells not expressing B7 onthe cell surface (CHO-B7⁻ cells) were used as controls. Before thestimulation, CHO cells were fixed with paraformaldehyde to decreasephosphotyrosine background. Previous studies have indicated that thistreatment leaves intact B7-CD28 interaction and the ensuing functionaleffects. See Gimmi, supra (1991). For the time zero point, lysis bufferwas added to the T cells first, immediately followed by addition of CHOcells to the mixture. CHO-B7⁺ cells induced specific tyrosinephosphorylation that was detected primarily on a substrate that migratedat 100 kD. The CHO-B7-induced tyrosine phosphorylation was detectablewithin 5 m of stimulation and remained elevated at plateau levels for atleast 30 m. CHO-B7-induced tyrosine phosphorylation was evident at avariety of CHO-T cell ratios, and has been consistently observed foronly the 100 kD substrate. CHO-B7⁻ cells did not induce tyrosinephosphorylation of pp100. The B7-induced tyrosine phosphorylation wasdependent upon CD28-B7 interaction as preincubation of CHO cells withanti-B7 mAb prevented CHO-B7 induced pp100 tyrosine phosphorylation.B7-CHO cells induced a slight increase in pp100 tyrosine phosphorylationin some experiments; however, this was not consistently observed.

[0158] In other experiments, alloantigen-induced T cell blasts weretested for CD28-induced tyrosine phosphorylation. T cells were culturefor 8 days with allogeneic irradiated cells and then stimulated withCD28 mAb. Tyrosine phosphorylation that was most pronounced on the 74and 100 kD substrates was observed. Thus, CD28 stimulation of T cellspreactivated with alloantigen, CD3 mAb, or PMA can induce tyrosinephosphorylation on a limited number of substrates that is early in onsetand brief in duration.

[0159] CD28-induced tyrosine phosphorylation prevented by CD45 and byherbimycin. Given that protein tyrosine kinase inhibitor herbimycin Acould efficiently inhibit CD28-induced IL-2 secretion, this inhibitorwas tested for effects on CD28-induced tyrosine phosphorylation. T cellswere treated overnight with PMA (5 ng/ml) in the presence of theindicated concentration of herbimycin A or in control medium. The cellswere collected, washed, and 8×10⁶ cells were stimulated with media orwith crosslinked anti-CD28 mAb for 2 m. Detergent-soluble proteins wereprocessed as previously described. Tyrosine phosphorylation induced byanti-CD28 mAb was nearly completely prevented in herbimycin-treatedcells under conditions that specifically inhibit CD28-induced IL-2production.

[0160] The brief temporal course of CD28 mAb-induced tyrosinephosphorylation suggested regulation by a phosphatase. To address theeffects of phosphatases on CD28-mediated signal transduction, T cellswere cultured overnight with PMA (5 ng/ml). 107 cells were incubated for10 m with media (control), biotinylated anti-CD45 mAb (9.4), anti-CD28mAb (9.3), or both. Monoclonal antibodies were crosslinked with avidinat time 0. The reaction was terminated after 2 m. Immunoblot analysiswith antiphosphotyrosine antibodies of detergent-soluble proteins wasperformed as previously described. CD28 crosslinking induced tyrosinephosphorylation on pp75 and pp100 that was completely prevented by CD45.Consistent with previous results described in Samelson, L. E. et al., J.Immunol., 145:2448 (1990), crosslinking of CD45 alone caused increasedtyrosine phosphorylation of a 120-135 kD substrate; this effect is alsoseen in CD28 plus CD45-treated cells. Thus, the above studies indicatethat CD28-induced tyrosine phosphorylation is sensitive to an inhibitorof src family protein tyrosine kinases, and furthermore, that the CD45protein tyrosine phosphatase can prevent CD28-induced protein tyrosinephosphorylation.

[0161] CTLA-4 expression predicts expression of IL-2 following CD28pathway activation. Purified resting T cells were stimulated withimmobilized anti-CD2 Ab, anti-CD3+mAb 9.3, PMA+ionomycin, PMA+mAb 9.3and PMA+ionomycin+mAb 9.3 in the presence or absence of theprotein-tyrosine kinase inhibitor herbimycin for 8 h. Duplicate Northernblots were hybridized to CTLA-4, CD28, IL-2 or HLA specific probes.Expression of CD28, CTLA-4 and IL-2 was then analyzed by Northern blot.IL-2 expression correlated well with CTLA-4 expression following CD28pathway activation. CTLA-4 and IL-2 expression were also suppressed to asimilar degree with herbimycin while CD28 expression remained unchanged.This suggests that the suppressive effects of protein-tyrosine kinaseinhibitors on CD28 pathway activation may be mediated throughsuppression of CTLA-4 expression.

SPECIFIC EXAMPLE XII Induction of MHC Independent T Cell Proliferationby CD28 and Staphylococcus Enterotoxins Protocol

[0162] Isolation of T cells. Peripheral blood was drawn from normalhuman volunteers. The mononuclear cell fraction was obtained by densitygradient centrifugation through a Ficoll-Hypaque (Pharmacia) cushion.This fraction was used in experiments utilizing peripheral bloodmononuclear cells (PBMCs). Purified resting T cells were obtained byincubating the mononuclear cells with an antibody cocktail directedagainst B cells, monocytes and activated T cells. The antibody coatedcells were then removed by incubation with goat anti-mouseimmunoglobulin-coated magnetic beads (Advanced Magnetics Inc.) aspreviously described in June, supra (1987). This method has routinelyyielded a population >99% CD2⁺ by flow cytometry.

[0163] Proliferation assays. Proliferation was measured by culturing5×10⁵ purified T cells or PBMC's in each well of a 96 well microtiterplate. The final culture volume was 200 μl of RPMI 1640 (Gibco)supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100μg/ml) and 2 Mm L-glutamine. Staphylococcal Enterotoxin A (SEA),Staphylococcal Enterotoxin B (SEB) (Toxin Technologies) and cyclosporineA (Sandoz) were added in the indicated doses at the initiation of theculture. Anti-CD28 monoclonal antibody (mAb 9.3, gift from J. Ledbetter)and anti-HLA-DR monoclonal antibody (mAb L243, gift from J. Ledbetter)were added at the start of the culture period. Tritiated thymidine(³H-TdR, ICN) was included at a concentration of 1 μCi per well for thefinal 8 h of the culture. The cells were harvested onto glass microfiberfilter strips (Whatman) after 72 h using a PHD cell harvester (CambridgeTechnologies) and counted on a liquid scintillation counter (LKB). Allvalues are expressed as the mean cpm±standard deviation of triplicate orquadruplicate cultures.

[0164] Flow Cytometry. One ml cultures of T cells were incubated withmedia alone, SEA (100 ng/ml) or SEB (1 μg/ml), SEA or SEB plus anti-CD28antibody (1 μg/ml) or PMA (3 ng/ml) plus anti-CD28 antibody (1 μg/ml) at37° C. for 72 h. Aliquots of each sample were stained with acridineorange (Polysciences) for cell cycle analysis as described inDarzynkiewicz, Z., Meth. Cell. Biol., 33:285 (1990), FITC conjugatedanti-IL2 receptor antibody (Coulter), FITC conjugated anti-HLA-DRantibody (Becton-Dickinson) or an isotype-matched irrelevant antibody(Becton-Dickinson). Each sample was analyzed on a FACScan flow cytometer(Becton-Dickinson).

Results

[0165] CD28 provides costimulatory activity for superantigen-activaedepurified T cells. Highly purified T cells were cultured with gradedconcentrations of either SEA (0.1 ng/ml to 1.0 μg/ml) or SEB (.01 μg/mlto 100 μg/ml). Replicate cultures were prepared in which a stimulatoryantibody to CD28 was added. The cultures were pulsed with ³H-TdR for thefinal 8 h of a 72 h culture and incorporated thymidine determined byliquid scintillation counting as described above. Each condition wasperformed in quadruplicate. Treatment with SEB alone failed to inducethymidine incorporation above control cultures. However, the addition ofanti-CD28 antibody resulted in significant proliferation to graded dosesof SEB. Treatment with anti-CD28 antibody alone had no effect. The lackof accessory cells was verified by an absence of proliferation to PHA.Identical results were obtained using SEA.

[0166] Stimulation with SEA or SEB leads to cell cycle entry. Since CD28stimulation alone does not induce T cell cycle entry, the observationthat CD28 provided costimulatory activity for T cells treated witheither SEA or SEB suggested that these enterotoxins could induce cellcycle entry in purified T cells. In order to examine this, purified Tcell cultures were stimulated with SEB (1 μg/ml) alone or with SEB (1μg/ml) plus anti-CD28 monoclonal antibody (1 μg/ml) for 48 h and stainedwith acridine orange for cell cycle analysis. Unstimulated cells wererun simultaneously in order to determine the G₀/G₁ interface. Those withan increased RNA content but unchanged DNA content were considered G₁phase cells. Cells with increases in both RNA and DNA content wereconsidered in S, G₂ or M phases. Concomitantly, aliquots were stainedwith FITC-conjugated anti-IL-2 receptor antibody. Treatment withenterotoxin alone for 48 h resulted in progression of greater than 10%of the T cells from G₀ to G₁ as determined by an increase in RNAstaining with no increase in DNA content. Similarly, enterotoxin aloneinduced IL-2 receptor expression in 15% of the cells at 72 h. Incontrast, when the anti-CD28 monoclonal antibody was present, asignificant proportion of the cells that had left the G₀ stage of thecell cycle were found to have increased their DNA content and thus arein either the S, G₂ or M phases of the cell cycle. These data indicatethat stimulation with SEB alone is sufficient to activate the T cell butdelivers an inadequate signal for complete progression through the cellcycle. Provision of a second signal by simultaneous stimulation of theCD28 pathway allowed the cell to progress to S-phase and proliferate.

[0167] Proliferation of T cells stimulated by SEA and anti-CD28 isresistant to cycl sporine A. CD28 has been shown to utilize a signaltransduction pathway that is resistant to the effects of cyclosporine A(CsA) when the initial signal is provided by PMA, and partiallyresistant to CsA when cells are initially activated through the T cellreceptor. See June, supra (1987). In order to further examine thepathways involved in T cell activation by enterotoxin, cyclosporine A (1μg/ml) was included in cultures activated by SEA and SEA plus anti-CD28antibody (³H-TdR incorporation determined as described above). As forSEB, SEA alone did not induce thymidine incorporation whereas additionof antibody against CD28 resulted in significant proliferation. Even inthe presence of cyclosporine A (1 μg/ml), there was a dose dependentincrease in proliferation when cultures were activated by a combinationof SEA and anti-CD28 antibody. Control cultures activated with PMA plusanti-CD28 antibody were resistant to cyclosporine A and activation byPMA plus ionomycin was sensitive to cyclosporine A.

[0168] Activation by SEB and anti-CD28 is independent of class II MHC.Previous work has demonstrated that the staphylococcal enterotoxins arecapable of simultaneously binding the TCR and class II MHC molecules onthe surface of antigen presenting cells (APCs). See Herrmann, T. et al.,Eur. J. Immunol., 19:2171 (1989); and Chintagumpala, M. M. et al., J.Immunol., 147:3876 (1991). The observation that proliferation was notobserved unless APCs were present in the culture was interpreted to meanthat T cell activation by superantigen is dependent upon class II MHCexpression as discussed in Fleischer, B. et al., J. Exp. Med., 167:1697(1988); Carlsson, R. et al., J. Immunol., 140:2484 (1988); and Herman,A. et al., J. Exp. Med., 172:709 (1990). Our observation that highlypurified T cells could be induced to proliferate by simultaneousstimulation with enterotoxin and anti-CD28 antibody suggested that classII MHC may not be absolutely required for superantigen activation of Tcells. Alternatively, activated T cells can express class II MHC andthus might provide class II-dependent superantigen presentation to otherT cells in trans.

[0169] To examine this possibility, a blocking antibody against HLA-DR,monoclonal antibody L243, was included in cultures of T cells and PBMCsactivated by enterotoxin or enterotoxin plus anti-CD28 antibody as shownin FIG. 16. Each point is expressed as the mean±the standard deviationof triplicate or quadruplicate cultures. No significant proliferationwas observed with SEB alone. As shown previously, inclusion of anti-CD28antibody allows SEB to induce T cell proliferation in a dose-dependentmanner. There was no decrease in proliferation when anti-class IIantibody was included in the cultures at doses of 1.0 or 10 μg/ml (datafor 10 μg/ml not shown). In contrast, as shown in FIG. 17, theproliferation of PBMCs isolated from the same donor and stimulated withenterotoxin was significantly inhibited by anti-class II antibody. Inaddition, HLA-DR expression at 24 and 72 h was examined by purified Tcells activated with SEA (0.1 or 1.0 ng/ml) with and without CD28costimulation. There was no expression of HLA-DR in either condition asdetermined by flow cytometry. This indicated that the T cellproliferation induced by enterotoxin+anti-CD28 is not dependent onpresentation by an MHC class II molecule.

SPECIFIC EXAMPLE XIII Prevention of Programmed Cell Death

[0170] A series of experiments were done to test whether anti-CD28 mAbmight prevent cell death in mature T cells. Jurkat leukemia cells arecommonly used as an example of mature T cells that mimic physiologiceffects found in peripheral blood T cells. For example, Jurkat cells canbe induced to secrete IL-2 with anti-CD3 mAb and anti-CD28 mAb, andJurkat cells can be infected and killed by HIV-1. The Jurkat lineJHMI-2.2 was obtained from A. Weiss (UCSF); the muscarinic M₁ receptorsubtype has been transfected and is stably expressed in these cells.JHMI-2.2 cells, 0.3×10⁶/well, were added to culture wells in completemedium, or to wells that contained plastic-adsorbed anti-CD3 mAb G19-4,in the presence or absence of 9.3 mAb 10 μg/ml, or 9.3 mAb alone. Celldeath was scored after 1 to 3 days of culture and graded as 0 (none), 1+(20 to 70% of cells dead), and 2+ (70 to 100% of cells dead), and wasdetermined by visual inspection of the wells, and confirmed by trypanblue permeability. As shown in Table 10, cells in medium continued togrow and remain viable while cells in anti-CD3-treated wells died. Incontrast, the cells in wells containing anti-CD3 plus anti-CD28continued to proliferate. The ability of anti-CD28 to rescue cells fromanti-CD3-induced cell death was specific, because carbacol (30 μM) (aspecific agonist of M₁ receptors that is believed to activate signaltransduction in cells via a mechanism distinct from the T cellreceptor/CD3 complex), also induced cell death in Jurkat cells.Anti-CD28 did not prevent carbacol-induced cell death. TABLE 10CONDITION CELL DEATH (GRADE 0-2) EXPERIMENT #1 Medium 0 anti-CD3   2+anti-CD3 + anti-CD28 0 EXPERIMENT #2 Medium 0 Carbacol   1+ Carbacol +anti-CD28   2+

SPECIFIC EXAMPLE XIV Bone Marrow Studies

[0171] Proliferation of T cells after activation of T cells with solubleor immobilized anti-CD3 (OKT3). A series of titration studies wereperformed using soluble or immobilized OKT3 to activate and induce Tcell proliferation. Immobilized OKT3 (2 μg/ml precoated plates for 1 hat 37° C.) and soluble OKT3 (10 ng/ml) consistently induced T cellproliferative responses from E-rosette (E⁺) purified T cells or PBL. PBLor purified T cells were activated by incubation for 1 h to 7 days onimmobilized OKT3 or by adding 10 ng/ml of soluble OKT3 at the beginningof culture. In a series of experiments, proliferation after activationof T cells with immobilized OKT3 was comparable to proliferativeresponses by PBL after activation with soluble OKT3.

[0172] Cytotoxicity mediated by anti-CD3 and anti-CD28 triggered PBL Thecytotoxicity of anti-CD3 activated PBL after 7 days of culture in thepresence of low doses of IL-2 or anti-CD28 was tested. In this set ofexperiments, the ability of CD28 to induce increases in lymphokineproduction to substitute for previously reported immune augmentedeffects of in vitro T cell treatment with IL-2 has been examined. Onelytic unit is equivalent to 20% lysis of 5×10³ target cells per 1×10⁶effector cells as discussed in Press, H. F. et al., J. Clin. Immunol.1:51-83 (1981). Various targets, including Daudi and K562, were tested.Cytotoxicity results of a representative experiment are shown in Table11. TABLE 11 CELL GROWTH Target - Daudi Target - K562 STIMULUS (LU) (LU)anti-CD3 plus IL-2 14.9 10.6 anti-CD3 plus anti-CD28 12.1 6.1

[0173] OKT3-induced cytotoxicity in PBL comparable to T cells. In 5different subjects, PBL were compared with T cells (E⁺) in their abilityto kill Daudi, K562, and BSB cells 8 days after being activated withOKT3. These experiments were performed in X-Vivo 10 supplemented with 5%human serum (HS). The mean cytotoxicity in 5 normal subjects using PBLdirected at Daudi, K562, and BSB were 15.0, 7.4, and 9.2 LU,respectively. In T cells from the same 5 subjects, the mean cytotoxicitydirected at Daudi, K562, and BSB were 14.3, 7.7, and 9.3 LU,respectively.

[0174] Cytotoxicity as a function of in vitro time in cell culture. Inorder to test for optimal cytotoxicity, T cells were cultured withanti-CD3 and IL-2 for 31 days and tested at weekly intervals forcytotoxicity against Daudi and K562. Logarithmic cell growth wasmaintained during this time, with a 300-fold expansion in cell number.Cytotoxicity was a strong function of culture duration however, with<0.1, 24, 3.5, 0.5, 1.0 LU at 0, 8, 15, 22, and 29 days of culture.Similar results were found when Daudi was the target, with <0.01, 23, 5,2, and 5 LU at 0, 8, 15, 22, and 29 days of culture.

[0175] Cytotoxicity induced by soluble or immobilized OKT3. In 7experiments, cytotoxicity mediated by T cells after activation wascompared with soluble or immobilized OKT3. Both methods inducedcytotoxicity directed at Daudi and K562. Soluble OKT3 activated T cellsmediated a mean cytotoxicity of 27 LU (SD-18) directed at Daudi and amean cytotoxicity of 21 LU (SD -16) directed at K562. Immobilized OKT3activated T cells mediated a mean cytotoxicity of 22 LU (SD-16) directedat Daudi and a mean cytotoxicity of 12 LU (SD-8) directed at K562. Theseexperiments were performed with E⁺ cells in RPMI 1640 supplemented with10% fetal bovine serum (FBS). Cytotoxicity was assessed 7 to 8 daysafter triggering with soluble (10 ng/ml) or immobilized (2 μg/ml) OKT3.

[0176] Eff cts f IL-2 concentrations. The dose of IL-2 was titratedafter establishing the optimal time of OKT3 activation. In severalexperiments, the doses of IL-2 were gradually reduced from 6000 IU/ml to60 IU/ml. Proliferation, as measured by tritiated thymidineincorporation after 3 days of culture, remained constant as IL-2 wastitrated in this range. In contrast, cytotoxicity varied with the doseof IL-2, and was maximal at lower doses of IL-2 (lytic units with Dauditargets were 28, 9, 8.5, 9 and 4.8 LU after culture in 30, 150, 300, 600and 6000 IU/ml of rIL-2). The data show that both proliferative andcytotoxic responses of the anti-CD3 triggered T cells can be obtainedand maintained in low doses of IL-2.

[0177] Effects of serum and medium on proliferation and cytotoxicity.The ability of HS, FBS, and serum free media were compared for theirability to support growth and maintain cytotoxicity. The data show thatproliferation and cytotoxicity directed at Daudi, K562, and BSB rapidlydecreased below a serum concentration of 2%. There were no significantdifferences between X-Vivo 10 and RPMI 1640.

[0178] Bone marrow mononuclear cells (BMMNC) as a source of CTC afteranti-CD3 and anti-CD28 treatment. To test whether BMMNC might serve as asource of T cells for therapy in patients with malignancies, BMMNC werecultured in X-Vivo 10 medium after OKT3 and IL-2 or anti-CD28stimulation. Although the BMMNC population initially contained only 25%CD3⁺ cells, proliferative and cytotoxic responses were excellent after 2weeks of culture. T cells expanded more than 40-fold after CD3 and IL-2stimulation and cytotoxicity was between 5 and 12 LU at 8 to 15 days ofculture when tested against Daudi and K562. These data show that BMMNCobtained from autologous bone marrow harvest from a patient before bonemarrow transplantation provide a suitable source of cytotoxic T cells.Table 12 shows that both normal and patient bone marrows providesatisfactory sources of cytotoxicity after CD3 and IL-2 treatment. In 4experiments using normal bone marrow or autologous bone marrow, therewas a median of 89-fold expansion of cells (range 18- to 173-fold) after9 to 19 days of culture. Mean cytotoxicity directed at Daudi and K562mediated by BMMNC stimulated with OKT3 was 5.5 LU (range 2-11) and 4.3LU (range 2-8), respectively. All cultures were tested 14 days afteractivation with OKT3 and expanded in the presence of 50 IU/ml of IL-2.Either soluble OKT3 10 ng/ml(s) or immobilized OKT3 (coated with 2μg/ml)(l) were added as indicated. TABLE 12 Daudi K562 Source OKT3 (S orI) Fold Increase LU LU PBL 1 I 47 6 3 2 I 4 11 11 3 S 12 9 6 4 S 176 5 4BMMNC 1 I 89 2 2 2 I 18 11 8 3 S 22 6 5 4 S 173 3 2

[0179] Anti-CD3 plus anti-CD28 treatment of BMMNC as a source ofeffector T cells. Proliferative and cytotoxic responses from 3 patientswere tested. The patients had received extensive chemotherapy and yettheir PBL or BMMNC maintained strong proliferative and cytotoxicresponses after anti-CD3 plus anti-CD28 treatment. PBL or BMMNC(1.5×10⁵) were cultured in RPMI plus 5% human serum in the presence ofimmobilized OKT3 mAb or 50 IU/ml rIL-2 or 9.3 mAb 0.5 μg/ml.Proliferation was assessed on day 3 of culture and cytotoxicity on day7. Table 13 summarizes the results for BMMNC. TABLE 13 Proliferation ³HCytotoxicity Cytotoxicity SAMPLE incorp. (cpm) Daudi (LU) K562 (LU) BoneMarrow #171 AML, Relapsed OKT3 23,479 — — OKT3 + anti-CD28 mAb 58,56318.0  4.8 OKT3 + IL-2 48,745 15.1  4.6 PBL P#632 non-Hodgkins lymphoma,pre-transplant OKT3 58,670 — — OKT3 + anti-CD28 mAb 77,046 14.8 12.7OKT3 + IL-2 63,603 18.8 18.1 PBL P#635 Hodgkins lymphoma, pre-transplantOKT3 27,758 — — OKT3 + anti-CD28 mAb 46,133 — — OKT3 + IL-2 43,918 — —

[0180] OKT3-activated T cells (CTC) do not inhibit hematopoieticprogenitor growth. In order to determine whether BMMNC mixed withOKT3-activated T cells (CTC) in hematopoietic progenitor assays wouldinhibit the development of CFU-GM, CTC obtained from PBL after a week ofgrowth was mixed with fresh BMMNC and plated the mixtures into theCFU-GM assay. The autologous CTC were mixed with BMMNC in variousratios, incubated for 1 h at 37°, and then plated in a standard CFU-GMassay. The CTC had no deleterious effect on colony formation, as thenumber of CFU-GM colonies was within 75% of control over a wide varietyCTC:BMMNC ratios (ratios of 1:25 to 5). The number of CFU-GM colonieswas not inhibited greater than 90% (an accepted % inhibition of CFU-GMin purged autologous marrow grafts) even at a ratio of 1 CTC to 1 BMMNC.These data suggest that CTC will not inhibit or delay engraftment ofautologous bone marrow transplants.

[0181] Expansion and cytotoxic functions of PBL or BMMNC from patientsbefore BMT. In order to determine whether PBL or BMMNC from patientsheavily pretreated for AML or lymphoma could be activated with anti-CD3and grown in low dose IL-2, PBL or BMMNC obtained prior to BMT onseveral patients was tested. The PBL and BMMNC of the patients testedproliferated and exhibited cytoxicity in a fashion comparable to thatseen in normal PBL or BMMNC obtained from normal allogeneic marrowdonors. It was anticipated that some patients that had been heavilytreated with chemotherapy or radiation would have low counts or havepoor responses to anti-CD3 activation. Thus, the number of startingcells was increased in the protocol to compensate for cell loss orinability to proliferate. The use of 9.3 as a costimulant to anti-CD3activated T cells to enhance helper activity or enhance cytotoxicitycould result in improved in vitro expansion of activated T. cells. Datapresented in this example (Specific Example XIV) show that mAb 9.3 cancorrect proliferative defects in post-transplant lymphocytes furthersupporting the rationale for using OKT3/9.3 costimulation approach toaccelerate immune reconstitution and enhance cytotoxicity directed atmallignant cells in ABMT recipients. A recent study by Katsanis, E. etal., Blood 78:1286-1291 (1991) shows that T cells from BMT recipientscan be expanded by stimulation with OKT3 and IL-2.

[0182] Messenger RNA levels for IL-2 receptors (IL-2R), IL-2, and IL-3in PBL from short and long-term BMT recipients. Earlier studies (seeLum, L. G. et al., Blood Suppl. (Abstract) (1991)) suggested that Tcells from BMT recipients fail to secrete IL-2 or express IL-2R. Suchdefects may be due to failure of mRNA synthesis for lymphokines orlymphokine receptors. A determination was made whether T cells from BMTpatients failed to express detectable levels of mRNA for IL-2, IL-2R andIL-3. PBL from 11 allogeneic (3 short-term, ST, and 8 long-term, LT) and4 autologous recipients (2 ST and 2 LT) were tested for levels of IL-2R,IL-2, and IL-3 mRNAs without stimulation (−) or after phytohemagglutinin(PHA) and phorbol ester (TPA) stimulation (+). cDNA synthesized byreverse transcriptase (RTase) from total RNA was amplified by PCR usingspecific primers and the PCR products run on 1.5% agarose gelscontaining ethidium bromide. Table 14 shows the fraction and percent ofrecipients whose PBL had detectable levels of mRNA for IL-2R, IL-2, andIL-3. TABLE 14 STIMULATION IL-2R (%) IL-2 (%) IL-3 (%) AllogeneicRecipients − 2/11 (18) 1/9 (11) 8/11 (73) + 9/11 (82) 8/9 (89) 7/10 (70)Autologous Recipients −  2/4 (50) 3/4 (75)  4/4 (100) +  4/4 (100) 3/4(75)  3/4 (75)

[0183] PBL from a high proportion of ST and LT autologous and allogeneicBMT recipients expressed levels of mRNAs for IL-2R, IL-2, and IL-3 afterstimulation with PHA+TPA. In the ST recipients tested, 2 of 2 ABMTrecipients and 3 of 3 allogeneic recipients tested had PBL thatexpressed mRNA levels of IL-2R and IL-3; 2 of 2 allogeneic recipientstested expressed mRNA for IL-2. In most cases, defective mRNA synthesisfor IL-2R, IL-2, and IL-3 may not be responsible for defects in IL-2secretion and IL-2R expression. Posttranscriptional events may play amore important role in defective lymphokine secretion by T cells fromBMT recipients.

[0184] CTC help Ig synthesis and express mRNA for lymphokines andperforin. As discussed in Ueda, M. et al., J. Cell. Biochem. (Abstract)(submitted 1992), helper activity was assessed by adding normal T cellsor T cells activated with OKT3 to normal B cells after PW stimulation asmeasured by an ELISA-Plaque (PFC) assay. The number of PFC per million Bcells cultured was 3200, 4100, 8800 when 25, 50 or 75×10³ normal T cellswere added. When the same numbers of CTC were added, the number of PFCwere 220, 2100, and 2600. Thus, CTC exhibit substantial helper activity.Furthermore, CTC did not suppress normal autologous or allogeneic T andB cells in a suppressor assay for Ig synthesis. Helper activity wasradioresistant. Messenger RNA for IL-2, IL-3, IL-6 and perforin wasdetected from 6 h to more than 3 days after OKT3 activation using aRtase-PCR method. In summary, CTC help B cells produced Ig and did notsuppress Ig synthesis by normal T and B cells. Thus, adoptive transferof CTC after BMT may not only mediate a GVL effect but may accelerateimmune reconstitution. patients or paired by adding anti-CD28. In vitrodata on anti-CD3 and anti-CD3/anti-CD28 stimulated proliferativeresponses of T cells from BMT recipients support the premise that usingMab 9.3 in combination with OKT3 may have potent in vivo clinicaleffects as reported. See Joshi, I. et al., Blood Suppl. (Abstract)(1991). T cells from BMT recipients have defects in proliferation aftermitogen or anti-CD3 stimulation. Previous studies show thatcostimulation of normal T cells with anti-CD3 (G19-4) and 9.3 enhanceanti-CD3-induced proliferation by stabilizing lymphokine mRNAs.Experimentation to assess the ability of anti-CD3 (G19-4 or OKT3)+9.3 tocorrect defective anti-CD3-induced proliferative responses in PBL fromautologous and allogeneic BMT recipients (53-605 days post BMT) wasperformed. PBL from recipients or controls were stimulated for 3 dayswith G19-4, G19-4+9.3, OKT3, or OKT3+9.3. 9.3 was added at a finalconcentration of 100 ng/ml. Fifteen tests were performed on ABMTrecipients and sixteen tests were performed on allogeneic recipients.Table 15 shows the number of recipients whose PBL increased (↑), ordecreased (↓), or did not change (⇄) their proliferative responses afterthe addition of 9.3 to anti-CD3 stimulated PBL. The parenthesis indicatepercent of recipients whose proliferative responses increased after theaddition of 9.3. TABLE 15 TREATMENT BTM RECIPIENTS CHANGE G19-4 + 9.3OKT3 + 9.3 Autologous BMT ↑  9 (60%) 9 (82%) ↓  3 (20%) 2 (18%) ⇄  3(20%) 0 (0%) Allogeneic BMT ↑ 11 (69%) 8 (62%) ↓  5 (31%) 5 (38%) ⇄  0(0%) 0 (0%)

[0185] Costimulation of G19-4+9.3 or OKT3+9.3 significantly increasedproliferative responses induced by G19-4 or OKT3 alone (p<0.05, pairedrank-sum) in ABMT recipients. In summary, defects in anti-CD3-induced Tcell proliferation in BMT recipients were repaired by costimulation with9.3. These findings have therapeutic implications for patients withimmune defects manifest with impaired T cell proliferation. Indeedsimilar results have been obtained which indicate that the proliferativedefect of T cells from patients with HIV infection can be repaired withanti-CD28 treatment. See Lane, H. C. et al., J. Engl. J. Med., 313:85(1985) regarding proliferate defects in HIV.

[0186] Costimulation with anti-CD3 OKT3 and anti-CD28 9.3 enhanceddectectable mRNA levels for IL-2 in PBL from ABMT recipient. PBL from ashort-term ABMT recipient were studied for expression of mRNA levels forIL-2 after stimulation with OKT3 and costimulation with OKT3/9.3 usingRTase-PCR. cDNA synthesized by RTase from total RNA was amplified by PCRusing specific primers for IL-2 and the PCR products run on 1.5% agarosegels containing ethidium bromide. Consistent with the findings in theprevious paragraph, activated T cells from the ABMT recipient did nothave detectable levels of mRNA for IL-2 after OKT3 stimulation alone,whereas the same T cells costimulated with OKT3/9.3 had a distinct bandfor IL-2 of 458 bp detected on ethidium bromide stained agarose gel.This is an example of how OKT3/9.3 costimulation can repair an apparentdefect in the expression of mRNA for IL-2 in T cells from ABMTrecipients.

[0187] Stimulation of negatively selected CD4⁺ cells with anti-CD28 9.3after activation with anti-CD3 induces IL-2 independent proliferativeresponses. CD4⁺ cells were purified by a series of negative selectionsteps as previously described in Thompson, C. B. et al., PNAS (USA)86:1333-1337 (1989). PBL were incubated with a cocktail of mAbs directedat non-CD28⁺ cells, washed, and incubated with immunomagnetic beadcoated with goat anti-mouse antibody. The CD28⁺ enriched cells werefurther purified by removing the CD8⁺ cells by treatment with anti-CD8and binding the CD8⁺ cells to the immunomagnetic beads.

[0188] The remaining CD28⁺, CD4⁺ T cells from a normal donor werecultured by adding cells to culture dishes containing plastic adsorbedOKT3. After 48 h, the cells were removed and placed in flasks containingeither rIL-2 (200 IU/ml) or anti-CD28 mAb (100 ng/ml). The cells werefed with fresh medium as required to maintain a cell density of0.5×10⁶/ml, and restimulated at approximately weekly intervals byculture on plastic adsorbed OKT3 for 24 h. The cells could be maintainedin logarithmic growth, with a 4 to 5 log₁o expansion in cells number. Asshown in FIG. 18, cells propagated with anti-CD3 and anti-CD28 routinelyexpanded 10 to 30-fold more than cells grown in optimal amounts ofanti-CD3 and IL-2. When synthetic medium (X-Vivo 10) not containing FBSwas used, anti-CD3 plus anti-CD28 treated cells also expanded 10-foldbetter than anti-CD3 plus IL-2 treated cells. The highly enriched CD4cells did not proliferate in the presence of optimal amounts of thelectin phytohemagglutinin (PHA). Thus, the in vitro expansion of CD4cells using anti-CD28 has an advantage over previously describedmethods, in that it is independent of the addition of exogenous growthfactors, as no IL-2 or any other growth factors were added to thesecells. In addition, this system does not require the presence ofaccessory cells, which is advantageous in clinical situations whereaccessory cells are limiting or defective.

[0189] Phenotypes of anti-CD3 activated T cells. Populations of CTCcells grown in IL-2 for 6 to 12 days contained predominantly CD3⁺ cells(greater than 84%, median 88%). The proportion of CD56⁺ cells (a markerfor NK cells) was less than 1.3%. Triggering of E⁺ cells with OKT3 ispreferentially selecting CD3⁺ cells. CD4⁺ cells were 18% or less andCD8⁺ cells were greater than 66%. Lytic activity did not correlate withthe proportions of CD56⁺ cells in the cultures.

[0190] Immunophenotype of T cells differs after anti-CD28 andIL-2-mediated cellular growth. To examine the subsets of T cells thatare expanded, PBL were propagated for 16 days using either anti-CD3 andIL-2 or anti-CD3 and anti-CD28. The percentage of CD4 and CD8 cells was23.8 and 84.5 in the cells grown in IL-2, and 56.0 and 52.6 in the cellsgrown in CD28. These results suggests that CD28 expansion favors theCD4⁺ cells, in contrast to the well established observation that CD8⁺cells predominate in cells grown in IL-2 (for example, see aboveparagraph; see also Cantrell, D. A. et al., J. Exp. Med. 158:1895(1983)). To further test this possibility, CD4 cells were enriched to98% purity using negative selection with monoclonal antibodies andmagnetic immunobeads as described elsewhere in this example. The cellswere cultured for one month using anti-CD3 and either IL-2 or anti-CD28to propagate the cells. There was equal expansion of the cells for thefirst 26 days of the culture, however, as can be seen in Table 16, thephenotype of cells diverged progressively with increasing time inculture. TABLE 16 CULTURE METHOD DAYS CD3 (%) CD4 (%) CD8 (%) anti-CD3 +IL-2 0 >99 >99 <1 6 >99 98 1 12 >99 85 10 20 >99 45 40 26 >99 12 78anti-CD3 + IL-2 0 >99 >99 <1 6 >99 >99 <1 12 >99 >99 <1 20 >99 >99 <126 >99 >99 <1

[0191] Use of anti-CD28 for in vitro expansion of TIL The use of IL-2and inactivated tumor cells to expand tumor infiltrating lymphocytes(TIL cells) for later adoptive immunotherapy with a variety of neoplasmshas demonstrated promise (e.g., see Rosenberg, S. A. et al., NEJM323:570-578 (1990)). TIL cells were isolated from a nephrectomy specimenfrom a patient with renal cell carcinoma. The cells were cultured withtumor cells, and either IL-2 or anti-CD28 and IL-2 with mAb OKT3 addedat weekly intervals, beginning at day 14. Table 17 demonstrates thatanti-CD28 is an improved method for the propagation of these cells insome patients, with a 20-fold greater yield of cells. Immunophenotypeanalysis also reveals that CD4 T cells are expanded in “TIL” cultures.Furthermore, these cells also exhibited potent cytotoxic activityagainst DAUDI targets, with 82.8, 69.7, 78.8 and 101.5 percent specificlysis at effector to target ratios of 40:1, 20:1, 10:1 and 5:1. TABLE 17Use of anti-CD28 to Expand TIL cells Tumor cells + IL-2 Tumor cells +IL-2 Day of Culture (1000 U/ml) anti-CD3, then anti-CD28  0 3.6 × 10⁷3.6 × 10⁷  5 5.8 × 10⁷ 1.3 × 10⁸ 12 1.7 × 10⁸ 1.4 × 10⁸ 16 2.0 × 10⁸ 2.6× 10⁸ 19 3.8 × 10⁸ 4.8 × 10⁸ 25 2.9 × 10⁸ 9.0 × 10⁸ 36 3.7 × 10⁸ 1.8 ×10⁹ 43 3.0 × 10⁸ 3.2 × 10⁹ 48 2.3 × 10⁸ 5.1 × 10⁹

[0192] Anti-CD3 and anti-CD28 costimulation enhances expression of mRNAfor IL-2 and TNF-α in CD4+ cells. To examine whether CD4 cellspropagated in vitro by anti-CD28 might be an effective source oflymphokines, resting CD4 cells were stimulated by anti-CD3 mAb for 48 hfollowed by the addition of 50 IU/ml of recombinant IL-2 and comparedwith CD4 T cells costimulated with anti-CD3 and anti-CD28. Total RNA washarvested from each combination. A total of 10 μg of RNA was loaded intoeach lane. A class I probe (HLA B7) was used to show uniform loading.The bolt was hybridized with ³²P labeled probes specific for IL-2, TNF-αand HLA in succession. On days 1 and 8, the cultures were restimulatedwith anti-CD3 mAb in the presence of IL-2 or anti-CD28 mAb 9.3 (0.1μg/ml). The blots were stripped, and rehybridized with a probe for aconstant region of HLA class I mRNA, to demonstrate equal loading of thelanes. As illustrated by the Northern Blot of FIG. 19, there was a clearenhancement of mRNA for IL-2 and TNF-α after costimulation with anti-CD3and anti-CD28 and the IL-2 and TNF-α mRNA exceeded that of the IL-2propagated cell by 10 to 50-fold. Similar results were obtained when theculture was examined for one month of culture, after weeklyrestimulation with anti-CD3 and IL-2 or anti-CD28.

[0193] Supernatants from long-term anti-CD3 and anti-CD28 stimulatedcultures of CD4⁺ cells contain substantial amounts of IL-2, GM-CSF, andTNF-α. After anti-CD-3 stimulation in the presence of 200 IU/ml of IL-2or the combination of anti-CD3 and anti-CD28 in the absence of IL-2,supernatants were tested for IL-2 content using the CTLL-2 cell line,GM-CSF content by ELISA, and TNF-α content by ELISA. The CD4⁺ cells wererestimulated with anti-CD3 and IL-2 or anti-CD3 and anti-CD28respectively at approximately weekly intervals. Anti-CD3 and anti-CD28cultures of CD4⁺ cells produced roughly the same amount of IL-2 found insupernatant obtained from anti-CD3 activated cells grown in exogenousIL-2. The amount of GM-CSF produced by anti-CD3 and anti-CD28 stimulatedCD4⁺ cells was also substantial. Although there were variations inlevels of TNF-α depending on when the supernatants were tested,costimulation with anti-CD3 and anti-CD28 was superior to stimulationwith anti-CD3 and IL-2 for inducing mRNA for TNF-α. These data indicatethat anti-CD28 costimulation with anti-CD3 may not only replace some ofthe functions of IL-2 but may enhance other synthetic functions of CD4⁺cells.

SPECIFIC EXAMPLE XV CD28 and CTLA-4 Expression Studies

[0194] CTLA-4 expression limited to CD28⁺ T cells. Utilizingsite-specific primers and DNA PCR of human genomic DNA, a 348 bpfragment corresponding to exon II of human CTLA-4 was generated, gelpurified and used as a ³²P-labeled probe. Purified human T cells wereseparated into CD28⁺ and CD28⁻ fractions by negative selection withmagnetic bead immunoabsorbtion. CD28⁺ T cells were either tested inmedia or stimulated with PMA+ionomycin or PMA+anti-CD28 mAb (mAb 9.3)for 12 h. CD28⁺ cells were stimulated with the last two conditions for12 h. RNA was extracted by guanidinium isothicyanate and purified overcesium chloride gradients. Equal amounts of RNA (as determined byethidium bromide staining) were loaded and separated on aformaldehyde-agarose gel and transferred to nitrocellulose todemonstrate equal loading of RNA. This blot was subsequently probed withthe CTLA-4 probe generated above. CTLA-4 was expressed in CD28⁺ cellsfollowing PMA or PMA+mAb 9.3 stimulation but not expressed in resting orstimulated CD28⁻ cells. The same blot was hybridized to a HLA probe toconfirm equal loading of RNA.

[0195] The expression of CTLA-4 induced under conditions causing CD28pathway activation. Purified resting CD28⁺ T cells were stimulated withPMA alone, ionomycin alone, and PMA+ionomycin for 1 h, 6 h, 12 h and 24h. RNA was extracted and analyzed by hybridization to CD28 and CTLA-4probes. Northern blot analysis showed that CTLA-4 expression was inducedby PMA or PMA+ionomycin, conditions which are costimulatory with CD28pathway activation. CTLA-4 expression was not induced by ionomycin,which is not costimulatory with CD28 pathway activation. In contrast,CD28 expression was constant with ionomycin or PMA and even appeared tobe suppressed with PMA and ionomycin stimulation. It should also benoted that the induction of CTLA-4 expression occurred as soon as 1 hafter stimulation, compared to 6-12 h with IL-2 expression followingCD28 pathway activation. Since expression of CTLA-4 precedes thebiological events caused by CD28 pathway activation (i.e. enhanced IL-2expression), CTLA-4 expression likely plays a role in the generation oflater events.

[0196] Purified human T cells were either untreated or stimulated for 1h, 4 h or 23 h with PMA+PHA, anti-CD28 mAb crosslinked with a secondantibody (goat anti-mouse Ig), or anti-CD5 mAb crosslinked in the samemanner. Northern blot analysis showed that crosslinking of CD28receptors, which also can activate the CD28 pathway through mechanismsdistinct from PMA and ionomycin, also induced CTLA-4 expression.

[0197] CD28⁺ cell lines slightly or not responsive to CD28 pathwayactivation do not express CTLA-4. Two cell lines that are CD28⁺ butresponded poorly (T cell line Jurkat C J) or not at all (myeloma cellline RPMI-8226) to CD28 costimulation as discussed in Kozbor, D. et al.,J. Immunol., 138:4128-4132 (1987) and Ledbetter, J. A. et al., PNAS(USA), 84:1384-1388 (1987), were stimulated with PMA+ionomycin+mAb 9.3and subsequently analyzed by Northern blot for CD28 and CTLA-4expression. Northern blot analysis of the T cell leukemia cell lineJurkat C J and of the myeloma cell line RPMI 8226 showed that these celllines did not express CTLA-4 despite CD28 expression.

[0198] T cells were incubated with various combinations of mitogensincluding phytohemagglutinin (PHA), phorbol myristate acetate (PMA), andionomycin (IONO), or anti-CD28 monoclonal antibodies (α-CD28), andexamined for the ability to induce CTLA-4 mRNA expression. As shown inFIG. 20, the combination of the mitogens PHA and PMA readily induceCTLA-4 expression in normal human T cells, but all combinations testedfailed to induce the expression of the CD28-isoform CTLA-4 in the JurkatT cell line. Both cell types express significant levels of CD28 mRNAunder all conditions tested.

[0199] Resting T cells were costimulated with anti-CD28 monoclonalantibodies to examine activation in normal human cells. As shown in FIG.21, normal human T cells stimulated by plate-adherent anti-CD3 (α-CD3)monoclonal antibodies at a concentration of 1 μg/ml induced only lowlevels of CTLA-4 mRNA expression first observable at 1 h afterstimulation. In contrast, costimulation of cells with anti-CD3monoclonal antibodies (1 μg/ml) soluble anti-CD28 (α-CD28) monoclonalantibody (1 μg/ml) led to a dramatic increase in the induction of CTLA-4mRNA expression.

SPECIFIC EXAMPLE XVI Efficacy of Administration of CTLA-4lg as aTreatment for Autoimmune Disease

[0200] Experimental Autoimmune Encephalomyelitis (EAE) is a rodent andprimate model for multiple sclerosis. Data has been generated on theeffect of administration of CTLA-4lg in both passive (indirect) andactive (direct) models of EAE. CTLA-4lg is a fusion protein consistingof the extracellular domain of human CTLA-4 fused to the constant regionof human IgG1 (referred to here as huCTLA-4lg).

[0201] Adoptively Transferred (Passive) EAE. In the passive EAE model,donor mice are immunized with 0.4 mg Myelin Basic Protein (MBP) inComplete Freund's Adjuvant (CFA), divided over four quadrants. Thedraining axillary and inguinal lymph nodes are removed eleven dayslater. Lymph node cells (4×10⁶/ml) are plated in 2 ml cultures in 24well plates, in the presence of 25 μg/ml MBP. After four days inculture, 30×10⁶ of the treated cells are injected into the tail vein ofeach naive, syngeneic recipient mouse.

[0202] The recipient mice develop a remitting, relapsing disease and areevaluated utilizing the following criteria:

[0203] 0—normal, healthy

[0204] 1—limp tail, incontinence; occasionally the first sign of thedisease is a “tilt”

[0205] 2—hind limb weakness, clumsiness

[0206] 3—mild paraparesis

[0207] 4—severe paraparesis

[0208] 5—quadriplegia

[0209] 6—death

[0210] Using this passive model of EAE, the effect of huCTLA-4lgtreatment of the donor cells on disease severity was tested in PLSJLF1/Jmice. Treatment of lymph node cells in vitro with MBP was performedeither in the presence or the absence of 30 μg/ml huCTLA-4lg. Thetreated cells were then introduced into a syngeneic recipient mouse. Asshown in FIG. 22, mice receiving huCTLA-4lg-treated cells (designatedPPIB CTLA-4) showed a significantly reduced severity of their firstepisode of disease as compared to mice receiving untreated cells(designated PPIA control). In addition, ensuing relapses in the micereceiving huCTLA-4lg-treated cells were less severe than in micereceiving cells not exposed to huCTLA-4lg. In fact, all five micereceiving huCTLA-4lg-treated cells stopped relapsing, and no longershowed signs of disease at 80-100 days post transfer.

[0211] Clinical disease severity was reduced even further by treatingboth the donor mice and the cultured cells with huCTLA-4lg (FIG. 23). Inthese experiments, donor mice of the SJL/J strain were given either 100μg huCTLA-4lg or 100 μg chimeric control IgG1 intraperitoneally each dayfor eleven days. T cells were then isolated from lymph nodes of thesedonors and cultured with MBP in vitro in the presence of either 30 μg/mlhuCTLA-4lg or chimeric control IgG1. The treated cells were thenintroduced into a syngeneic recipient. Treatment of either the donormice or the in vitro cultures resulted in significantly reduced clinicaldisease severity. Treatment of both the donor mice and the culturedcells with huCTLA-4lg was the most effective protocol for reducingclinical disease severity.

[0212] Direct administration of huCTLA-4lg to mice receiving adoptivelytransferred cells was also examined. As shown in FIG. 24, when PLSJLFI/Jrecipient mice were given 100 μg of either huCTLA-4lg or human IgG inPBS intraperitoneally on days 1 to 9 post transfer, no difference indisease severity Was observed between the two groups of mice. However,in experiments utilizing SJL/J mice, reduced disease severity duringrelapse was noted in mice treated with 100 μg huCTLA-4lgintraperitoneally on days 1 to 5 post transfer (FIG. 25). Ongoingexperiments are examining the effect of administration of a single doseof huCTLA-4lg to SJL/J recipient mice on day 2 post transfer. Table 18shows results of such an experiment, compared to the results obtainedwhen recipient mice are given either huCTLA-4lg or human IgG1 on days 1to 5 post transfer. While severity of the first episode of disease didnot appear to be significantly altered by treatment with huCTLA-4lgeither on day 2 or days 1-5, the duration of the first episode ofdisease was shorter for mice given huCTLA-4lg treatment (Table 18). Inaddition, huCTLA-4lg treatment on days 1-5 resulted in delayed onset ofthe first episode of disease. TABLE 18 Effect of Administration ofCTLA-4Ig to Mice with Adoptively Transferred EAE CTLA-4Ig CTLA-4Ig IgGControl (day 2) (days 1-5) (days 1-5) Mean day of onset 11.3 16.3 10.8Maximum score of first 3.2 2.8 2.8 episode Maximum score to date 3.5 3.23.0 (day 35) Average of first episode 5.2 6.7 9.8 (days)

[0213] Direct (Active) Model of EAE. Studies using a direct (active)model of EAE have also been conducted. In these experiments, huCTLA-4lgwas directly administered to mice immunized with MBP and treated withpertussis toxin (PT). PLSJLFI/J mice immunized with MBP on day 0 andinjected with PT intravenously on days 0 and 2 were given eitherhuCTLA-4lg or control IgG1 on days 0 to 7. Five of ten mice givenhuCTLA-4lg died on days 5 to 7, for reasons related to the PTadministered, and not the experimental design. The results of theexperiment for the remaining five mice are as shown in FIG. 27.Administration of huCTLA-4lg markedly reduced the mean clinical severityof disease in these animals, as compared to the mice treated with IgG1.These findings indicate that direct administration of soluble humanhuCTLA-4lg can provide an effective therapeutic strategy in thetreatment of autoimmune disease.

[0214] It should be appreciated that a latitude of modification, changeor substitution is intended in the foregoing disclosure and,accordingly, it is appropriate that the appended claims be construedbroadly and in a manner consistent with the spirit and scope of theinvention herein.

[0215] All publications and applications cited herein are incorporatedby reference.

What is claimed is:
 1. A method of inhibiting CD28 pathway activationassociated with an increase in cellular production of a T_(H)CD28lymphokine in a T cell population, wherein activation occurs by thebinding of a stimulatory CD28 ligand to a CD28 receptor stimulatorybinding site, the method comprising the steps of: a) selecting aninhibitory ligand capable of binding to the stimulatory CD28 ligand; b)providing the inhibitory ligand in a biologically compatible form; andc) administering the inhibitory ligand to the population in an amountsufficient to bind and inhibit the stimulatory ligand from binding theCD28 receptor stimulatory binding site.
 2. The method of claim 1,wherein the stimulatory ligand comprises a natural CD28 ligand.
 3. Themethod of claim 1, wherein the inhibitory ligand comprises an antibodyor fragment thereof to the stimulatory ligand.
 4. The method of claim 1,wherein the inhibitory ligand comprises a soluble form of CTLA-4.
 5. Themethod of claim 4, wherein the ligand comprises CTLA-4lg.
 6. The methodof claim 1, wherein the inhibitory ligand is of synthetic origin.
 7. Themethod of claim 1, wherein the inhibitory ligand comprises a recombinantmolecule.
 8. The method of claim 1, further comprising the step of: d)administering a second inhibitory ligand capable of binding but notstimulating the CD28 receptor binding site.
 9. A method of suppressingthe production of a T_(H)CD28 lymphokine by a population of T cells, themethod comprising the steps of: a) administering an inhibitory ligandwhich binds a stimulatory ligand for CD28; b) providing the ligand inbiologically compatible form; and c) administering the provided ligandin an amount sufficient to suppress production of the lymphokine in thepopulation.
 10. The method of claim 9, wherein the inhibitory ligandcomprises a soluble form of CTLA-4.
 11. The method of claim 10, whereinthe inhibitory ligand comprises CTLA-4lg.
 12. The method of claim 9,wherein the T cell population is in a patient in an autoimmune state.13. A method of suppressing T_(H)CD28 lymphokine production in a patienthaving a population of T cells, the method comprising the steps of: a)providing an inhibitory ligand which binds a natural stimulatory ligandfor CD28; and b) administering the inhibitory ligand in atherapeutically effective amount to the population of T cells.
 14. Themethod of claim 13, wherein the administration of the ligand to thepopulation of T cells is in vivo.
 15. The method of claim 13, whereinthe administration of the ligand to the population of T cells is invitro, and further comprising the step of: d) introducing the populationof T cells into the patient after administration.
 16. The method ofclaim 15, wherein the T cell population is removed from the patientprior to ligand administration.
 17. The method of claim 13, wherein theinhibitory ligand comprises a soluble form of CTLA-4.
 18. The method ofclaim 17, wherein the inhibitory ligand comprises CTLA-4lg.
 19. A methodof treating an autoimmune disease in a patient comprising the steps of:a) selecting an inhibitory ligand which binds a natural stimulatoryligand to CD28; and b) administering atherapeutically effective amountof the ligand to the patient.
 20. The method of claim 19, wherein thestimulatory ligand is B7 and the inhibitory ligand comprises a solubleform of CTLA-4.
 21. The method of claim 19, wherein the inhibitoryligand comprises CTLA-4lg.
 22. The method of claim 20, wherein theadministration is in vivo.
 23. The method of claim 20, wherein theadministration is in vitro to a population of cells removed from thepatient, and further comprising the step of: c) reintroducing the cellsto the patient after administration.
 24. The method of claim 20, whereinthe autoimune disease is multiple sclerosis.